Nucleotide sequences and corresponding polypeptides conferring modulated plant growth rate and biomass in plants

ABSTRACT

The present invention relates to isolated nucleic acid molecules and their corresponding encoded polypeptides able confer the trait of modulated plant size, vegetative growth, organ number, plant architecture, growth rate, seedling vigor and/or biomass in plants. The present invention further relates to the use of these nucleic acid molecules and polypeptides in making transgenic plants, plant cells, plant materials or seeds of a plant having plant size, vegetative growth, organ number, plant architecture, growth rate, seedling vigor and/or biomass that are altered with respect to wild type plants grown under similar conditions

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation-In-Part of co-pending applicationSer. No. 11/172,740 filed on Jun. 30, 2005, the entire contents of whichare hereby incorporated by reference and for which priority is claimedunder 35 U.S.C. § 120.

FIELD OF THE INVENTION

The present invention relates to isolated nucleic acid molecules andtheir corresponding encoded polypeptides able to modulate plant growthrate, vegetative growth, organ size, architecture seedling vigor and/orbiomass in plants. The present invention further relates to using thenucleic acid molecules and polypeptides to make transgenic plants, plantcells, plant materials or seeds of a plant having modulated growth rate,vegetative growth, organ number, architecture, seedling vigor and/orbiomass as compared to wild-type plants grown under similar conditions.

BACKGROUND OF THE INVENTION

Plants specifically improved for agriculture, horticulture, biomassconversion, and other industries (e.g. paper industry, plants asproduction factories for proteins or other compounds) can be obtainedusing molecular technologies. As an example, great agronomic value canresult from modulating the size of a plant as a whole or of any of itsorgans or the number of any of its organs.

Similarly, modulation of the size and stature of an entire plant, or aparticular portion of a plant, or growth rate, or seedling vigor allowsproduction of plants better suited for a particular industry. Forexample, reductions in the height of specific crops and tree species canbe beneficial by allowing easier harvesting. Alternatively, increasingheight, thickness or organ size, organ number may be beneficial byproviding more biomass useful for processing into food, feed, fuelsand/or chemicals (see the US Department of Energy website for EnergyEfficiency and Renewable Energy). Other examples of commerciallydesirable traits include increasing the length of the floral stems ofcut flowers, increasing or altering leaf size and shape or enhancing thesize of seeds and/or fruits. Changes in organ size, organ number andbiomass also result in changes in the mass of constituent molecules suchas secondary products and convert the plants into factories for thesecompounds.

Availability and maintenance of a reproducible stream of food and animalfeed to feed animals and people has been a high priority throughout thehistory of human civilization and lies at the origin of agriculture.Specialists and researchers in the fields of agronomy science,agriculture, crop science, horticulture, and forest science are eventoday constantly striving to find and produce plants with an increasedgrowth potential to feed an increasing world population and to guaranteea supply of reproducible raw materials. The robust level of research inthese fields of science indicates the level of importance leaders inevery geographic environment and climate around the world place onproviding sustainable sources of food, feed, chemicals and energy forthe population.

Manipulation of crop performance has been accomplished conventionallyfor centuries through plant breeding. The breeding process is, however,both time-consuming and labor-intensive. Furthermore, appropriatebreeding programs must be specially designed for each relevant plantspecies.

On the other hand, great progress has been made in using moleculargenetics approaches to manipulate plants to provide better crops.Through introduction and expression of recombinant nucleic acidmolecules in plants, researchers are now poised to provide the communitywith plant species tailored to grow more efficiently and produce moreproduct despite unique geographic and/or climatic environments. Thesenew approaches have the additional advantage of not being limited to oneplant species, but instead being applicable to multiple different plantspecies (Zhang et al. (2004) Plant Physiol. 135:615).

Despite this progress, today there continues to be a great need forgenerally applicable processes that improve forest or agricultural plantgrowth to suit particular needs depending on specific environmentalconditions. To this end, the present invention is directed toadvantageously manipulating plant size, organ number, plant growth rate,plant architecture and/or biomass to maximize the benefits of variouscrops depending on the benefit sought and the particular environment inwhich the crop must grow, characterized by expression of recombinant DNAmolecules in plants. These molecules may be from the plant itself, andsimply expressed at a higher or lower level, or the molecules may befrom different plant species.

SUMMARY OF THE INVENTION

The present invention, therefore, relates to isolated nucleic acidmolecules and polypeptides and their use in making transgenic plants,plant cells, plant materials or seeds of plants having life cycles,particularly plant size, vegetative growth, plant growth rate, organnumber, plant architecture and/or biomass, that are altered with respectto wild-type plants grown under similar or identical conditions.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Amino acid sequence alignment of homologues of Leads 80, 81,113, 114, ME08328, ME01905, ME01770, ME20023 (Clone 18200) and ME21445,SEQ ID NOS. 95, 97, 91, 83, 89, 85, 87, 93, 81. Conserved regions areenclosed in a box. A consensus sequence is shown below the alignment.

DETAILED DESCRIPTION OF THE INVENTION

1. The Invention

The invention of the present application may be described by, but notnecessarily limited to, the following exemplary embodiments.

The present invention discloses novel isolated nucleic acid molecules,nucleic acid molecules that interfere with these nucleic acid molecules,nucleic acid molecules that hybridize to these nucleic acid molecules,and isolated nucleic acid molecules that encode the same protein due tothe degeneracy of the DNA code. Additional embodiments of the presentapplication further include the polypeptides encoded by the isolatednucleic acid molecules of the present invention.

More particularly, the nucleic acid molecules of the present inventioncomprise: (a) a nucleotide sequence encoding an amino acid sequence thatis at least 85% identical to any one of Leads 80, 81, 113, 114, ME08328,ME01905, ME01770, ME21445 and ME20023, corresponding to SEQ ID Nos. 94,96, 90, 82, 88, 84, 86, 92, 80, respectively, (b) a nucleotide sequencethat is complementary to any one of the nucleotide sequences accordingto (a), (c) a nucleotide sequence according to any one of SEQ ID Nos.94, 96, 90, 82, 88, 84, 86, 92, 80, (d) a nucleotide sequence that is inreverse order of any one of the nucleotide sequences according to (c)when read in the 5′ to 3′ direction, (e) a nucleotide sequence able tointerfere with any one of the nucleotide sequences according to (a), (f)a nucleotide sequence able to form a hybridized nucleic acid duplex withthe nucleic acid according to any one of paragraphs (a)-(e) at atemperature from about 40° C. to about 48° C. below a meltingtemperature of the hybridized nucleic acid duplex, and (g) a nucleotidesequence encoding any one of amino acid sequences of Leads 80, 81, 113,114, ME08328, ME01905, ME01770, ME21445 and ME20023, corresponding toSEQ ID NOS. 95, 97, 91, 83, 89, 85, 87, 93, 81, respectively.

Additional embodiments of the present invention include thosepolypeptide and nucleic acid molecule sequences disclosed in SEQ ID NOS.94, 95, 96, 97, 90, 91, 82, 83, 88, 89, 84, 85, 86, 87, 92, 93, 80, 81.

The present invention further embodies a vector comprising a firstnucleic acid having a nucleotide sequence encoding a plant transcriptionand/or translation signal, and a second nucleic acid having a nucleotidesequence according to the isolated nucleic acid molecules of the presentinvention. More particularly, the first and second nucleic acids may beoperably linked. Even more particularly, the second nucleic acid may beendogenous to a first organism, and any other nucleic acid in the vectormay be endogenous to a second organism. Most particularly, the first andsecond organisms may be different species.

In a further embodiment of the present invention, a host cell maycomprise an isolated nucleic acid molecule according to the presentinvention. More particularly, the isolated nucleic acid molecule of thepresent invention found in the host cell of the present invention may beendogenous to a first organism and may be flanked by nucleotidesequences endogenous to a second organism. Further, the first and secondorganisms may be different species. Even more particularly, the hostcell of the present invention may comprise a vector according to thepresent invention, which itself comprises nucleic acid moleculesaccording to those of the present invention.

In another embodiment of the present invention, the isolatedpolypeptides of the present invention may additionally comprise aminoacid sequences that are at least 85% identical to any one of Leads 80,81, 113, 114, ME08328, ME01905, ME01770, ME21445 and ME20023,corresponding to SEQ ID Nos. ID NOS. 95, 97, 91, 83, 89, 85, 87, 93, 81,respectively.

Other embodiments of the present invention include methods ofintroducing an isolated nucleic acid of the present invention into ahost cell. More particularly, an isolated nucleic acid molecule of thepresent invention may be contacted to a host cell under conditionsallowing transport of the isolated nucleic acid into the host cell. Evenmore particularly, a vector as described in a previous embodiment of thepresent invention, may be introduced into a host cell by the samemethod.

Methods of detection are also available as embodiments of the presentinvention. Particularly, methods for detecting a nucleic acid moleculeaccording to the present invention in a sample. More particularly, theisolated nucleic acid molecule according to the present invention may becontacted with a sample under conditions that permit a comparison of thenucleotide sequence of the isolated nucleic acid molecule with anucleotide sequence of nucleic acid in the sample. The results of suchan analysis may then be considered to determine whether the isolatednucleic acid molecule of the present invention is detectable andtherefore present within the sample.

A further embodiment of the present invention comprises a plant, plantcell, plant material or seeds of plants comprising an isolated nucleicacid molecule and/or vector of the present invention. More particularly,the isolated nucleic acid molecule of the present invention may beexogenous to the plant, plant cell, plant material or seed of a plant.

A further embodiment of the present invention includes a plantregenerated from a plant cell or seed according to the presentinvention. More particularly, the plant, or plants derived from theplant, plant cell, plant material or seeds of a plant of the presentinvention preferably has increased size (in whole or in part), increasedvegetative growth, increased organ number and/or increased biomass(sometimes hereinafter collectively referred to as increased biomass),lethality, sterility or ornamental characteristics as compared to awild-type plant cultivated under identical conditions. Furthermore, thetransgenic plant may comprise a first isolated nucleic acid molecule ofthe present invention, which encodes a protein involved in modulatinggrowth and phenotype characteristics, and a second isolated nucleic acidmolecule which encodes a promoter capable of driving expression inplants, wherein the growth and phenotype modulating component and thepromoter are operably linked. More preferably, the first isolatednucleic acid may be mis-expressed in the transgenic plant of the presentinvention, and the transgenic plant exhibits modulated characteristicsas compared to a progenitor plant devoid of the polynucleotide, when thetransgenic plant and the progenitor plant are cultivated under identicalenvironmental conditions. In another embodiment of the present inventionthe modulated growth and phenotype characteristics may be due to theinactivation of a particular sequence, using for example an interferingRNA.

A further embodiment consists of a plant, plant cell, plant material orseed of a plant according to the present invention which comprises anisolated nucleic acid molecule of the present invention, wherein theplant, or plants derived from the plant, plant cell, plant material orseed of a plant, has the modulated growth and phenotype characteristicsas compared to a wild-type plant cultivated under identical conditions.

The polynucleotide conferring increased biomass or vigor may bemis-expressed in the transgenic plant of the present invention, and thetransgenic plant exhibits an increased biomass or vigor as compared to aprogenitor plant devoid of the polynucleotide, when the transgenic plantand the progenitor plant are cultivated under identical environmentalconditions. In another embodiment of the present invention increasedbiomass or vigor phenotype may be due to the inactivation of aparticular sequence, using for example an interfering RNA.

Another embodiment consists of a plant, plant cell, plant material orseed of a plant according to the present invention which comprises anisolated nucleic acid molecule of the present invention, wherein theplant, or plants derived from the plant, plant cell, plant material orseed of a plant, has increased biomass or vigor as compared to awild-type plant cultivated under identical conditions.

Another embodiment of the present invention includes methods ofenhancing biomass or vigor in plants. More particularly, these methodscomprise transforming a plant with an isolated nucleic acid moleculeaccording to the present invention. Preferably, the method is a methodof enhancing biomass or vigor in the transformed plant, whereby theplant is transformed with a nucleic acid molecule encoding thepolypeptide of the present invention.

Polypeptides of the present invention include consensus sequences. Theconsensus sequence is shown in FIG. 1.

2. Definitions

The following terms are utilized throughout this application:

Biomass: As used herein, “biomass” refers to useful biological materialincluding a product of interest, which material is to be collected andis intended for further processing to isolate or concentrate the productof interest. “Biomass” may comprise the fruit or parts of it or seeds,leaves, or stems or roots where these are the parts of the plant thatare of particular interest for the industrial purpose. “Biomass”, as itrefers to plant material, includes any structure or structures of aplant that contain or represent the product of interest.

Transformation: Examples of means by which this can be accomplished aredescribed below and include Agrobacterium-mediated transformation (ofdicots (Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson andLipman (1988) Proc. Natl. Acad. Sci. (USA) 85: 2444), of monocots(Yamauchi et al. (1996) Plant Mol. Biol. 30:321-9; Xu et al. (1995)Plant Mol. Biol. 27:237; Yamamoto et al. (1991) Plant Cell 3:371), andbiolistic methods (P. Tijessen, “Hybridization with Nucleic Acid Probes”In Laboratory Techniques in Biochemistry and Molecular Biology, P. C.vand der Vliet, ed., c. 1993 by Elsevier, Amsterdam), electroporation,in planta techniques, and the like. Such a plant containing theexogenous nucleic acid is referred to here as a T₀ for the primarytransgenic plant and T₁ for the first generation.

Functionally Comparable Proteins or Functional Homologs: This termdescribes those proteins that have at least one functionalcharacteristic in common. Such characteristics include sequencesimilarity, biochemical activity, transcriptional pattern similarity andphenotypic activity. Typically, the functionally comparable proteinsshare some sequence similarity or at least one biochemical. Within thisdefinition, analogs are considered to be functionally comparable. Inaddition, functionally comparable proteins generally share at least onebiochemical and/or phenotypic activity.

Functionally comparable proteins will give rise to the samecharacteristic to a similar, but not necessarily the same, degree.Typically, comparable proteins give the same characteristics where thequantitative measurement due to one of the comparables is at least 20%of the other; more typically, between 30 to 40%; even more typically,between 50-60%; even more typically between 70 to 80%; even moretypically between 90 to 100% of the other.

Heterologous sequences: “Heterologous sequences” are those that are notoperatively linked or are not contiguous to each other in nature. Forexample, a promoter from corn is considered heterologous to anArabidopsis coding region sequence. Also, a promoter from a geneencoding a growth factor from corn is considered heterologous to asequence encoding the corn receptor for the growth factor. Regulatoryelement sequences, such as UTRs or 3′ end termination sequences that donot originate in nature from the same gene as the coding sequence, areconsidered heterologous to said coding sequence. Elements operativelylinked in nature and contiguous to each other are not heterologous toeach other. On the other hand, these same elements remain operativelylinked but become heterologous if other filler sequence is placedbetween them. Thus, the promoter and coding sequences of a corn geneexpressing an amino acid transporter are not heterologous to each other,but the promoter and coding sequence of a corn gene operatively linkedin a novel manner are heterologous.

Misexpression: The term “misexpression” refers to an increase or adecrease in the transcription of a coding region into a complementaryRNA sequence as compared to the wild-type. This term also encompassesexpression and/or translation of a gene or coding region or inhibitionof such transcription and/or translation for a different time period ascompared to the wild-type and/or from a non-natural location within theplant genome, including a gene or coding region from a different plantspecies or from a non-plant organism.

Percentage of sequence identity: As used herein, the term “percentsequence identity” refers to the degree of identity between any givenquery sequence and a subject sequence. A query nucleic acid or aminoacid sequence is aligned to one or more subject nucleic acid or aminoacid sequences using the computer program ClustalW (version 1.83,default parameters), which allows alignments of nucleic acid or proteinsequences to be carried out across their entire length (globalalignment).

ClustalW calculates the best match between a query and one or moresubject sequences, and aligns them so that identities, similarities anddifferences can be determined. Gaps of one or more residues can beinserted into a query sequence, a subject sequence, or both, to maximizesequence alignments. For fast pairwise alignment of nucleic acidsequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For multiple alignment of nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The output is a sequencealignment that reflects the relationship between sequences. ClustalW canbe run, for example, at the Baylor College of Medicine Search Launcherwebsite and at the European Bioinformatics Institute website on theWorld Wide Web.

In case of the functional homolog searches, to ensure a subject sequencehaving the same function as the query sequence, the alignment has to bealong at least 80% of the length of the query sequence so that themajority of the query sequence is covered by the subject sequence. Todetermine a percent identity between a query sequence and a subjectsequence, ClustalW divides the number of identities in the bestalignment by the number of residues compared (gap positions areexcluded), and multiplies the result by 100. The output is the percentidentity of the subject sequence with respect to the query sequence. Itis noted that the percent identity value can be rounded to the nearesttenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to78.2.

Regulatory Regions: The term “regulatory region” refers to nucleotidesequences that, when operably linked to a sequence, influencetranscription initiation or translation initiation or transcriptiontermination of said sequence and the rate of said processes, and/orstability and/or mobility of a transcription or translation product. Asused herein, the term “operably linked” refers to positioning of aregulatory region and said sequence to enable said influence. Regulatoryregions include, without limitation, promoter sequences, enhancersequences, response elements, protein recognition sites, inducibleelements, protein binding sequences, 5′ and 3′ untranslated regions(UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, and introns. Regulatory regions can beclassified in two categories, promoters and other regulatory regions.

Seedling vigor: As used herein, “seedling vigor” refers to the plantcharacteristic whereby the plant emerges from soil faster, has anincreased germination rate (i.e., germinates faster), has faster andlarger seedling growth and/or germinates faster under cold conditions ascompared to the wild type or control under similar conditions. Seedlingvigor has often been defined to comprise the seed properties thatdetermine “the potential for rapid, uniform emergence and development ofnormal seedlings under a wide range of field conditions”.

Stringency: “Stringency,” as used herein is a function of nucleic acidmolecule probe length, nucleic acid molecule probe composition (G+Ccontent), salt concentration, organic solvent concentration andtemperature of hybridization and/or wash conditions. Stringency istypically measured by the parameter T_(m), which is the temperature atwhich 50% of the complementary nucleic acid molecules in thehybridization assay are hybridized, in terms of a temperaturedifferential from T_(m). High stringency conditions are those providinga condition of T_(m)-5° C. to T_(m)-10° C. Medium or moderate stringencyconditions are those providing T_(m)-20° C. to T_(m)-29° C. Lowstringency conditions are those providing a condition of T_(m)-40° C. toT_(m)-48° C. The relationship between hybridization conditions and T_(m)(in ° C.) is expressed in the mathematical equation:T_(m)=81.5-16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N)  (I)where N is the number of nucleotides of the nucleic acid molecule probe.This equation works well for probes 14 to 70 nucleotides in length thatare identical to the target sequence. The equation below, for T_(m) ofDNA-DNA hybrids, is useful for probes having lengths in the range of 50to greater than 500 nucleotides, and for conditions that include anorganic solvent (formamide):T_(m)=81.5+16.6 log {[Na⁺]/(1+0.7[Na⁺])}+0.41(% G+C)−500/L 0.63(%formamide)  (II)where L represents the number of nucleotides in the probe in the hybrid(21). The T_(m) of Equation II is affected by the nature of the hybrid:for DNA-RNA hybrids, T_(m) is 10-15° C. higher than calculated; forRNA-RNA hybrids, T_(m) is 20-25° C. higher. Because the T_(m) decreasesabout 1° C. for each 1% decrease in homology when a long probe is used(Frischauf et al. (1983) J. Mol Biol, 170: 827-842), stringencyconditions can be adjusted to favor detection of identical genes orrelated family members.

Equation II is derived assuming the reaction is at equilibrium.Therefore, hybridizations according to the present invention are mostpreferably performed under conditions of probe excess and allowingsufficient time to achieve equilibrium. The time required to reachequilibrium can be shortened by using a hybridization buffer thatincludes a hybridization accelerator such as dextran sulfate or anotherhigh volume polymer.

Stringency can be controlled during the hybridization reaction, or afterhybridization has occurred, by altering the salt and temperatureconditions of the wash solutions. The formulas shown above are equallyvalid when used to compute the stringency of a wash solution. Preferredwash solution stringencies lie within the ranges stated above; highstringency is 5-8° C. below T_(m) medium or moderate stringency is26-29° C. below T_(m) and low stringency is 45-48° C. below T_(m).

T₀: The term “T₀” refers to the whole plant, explant or callus tissue,inoculated with the transformation medium.

T₁: The term T₁ refers to either the progeny of the To plant, in thecase of whole-plant transformation, or the regenerated seedling in thecase of explant or callous tissue transformation.

T₂: The term T₂ refers to the progeny of the T₁ plant. T₂ progeny arethe result of self-fertilization or cross-pollination of a T₁ plant.

T₃: The term T₃ refers to second generation progeny of the plant that isthe direct result of a transformation experiment. T₃ progeny are theresult of self-fertilization or cross-pollination of a T₂ plant.

3. Important Characteristics of the Polynuceotides and Polypeptides ofthe Invention

The nucleic acid molecules and polypeptides of the present invention areof interest because when the nucleic acid molecules are mis-expressed(i.e., when expressed at a non-natural location or in an increased ordecreased amount relative to wild-type) they produce plants that exhibitmodulated biomass, growth rate, or seedling vigor as compared towild-type plants, as evidenced by the results of various experimentsdisclosed below. This trait can be used to exploit or maximize plantproducts. For example, the nucleic acid molecules and polypeptides ofthe present invention are used to increase the expression of genes thatcause the plant to have modulated biomass, growth rate or seedlingvigor.

Because the disclosed sequences and methods increase vegetative growth,and growth rate, the disclosed methods can be used to enhance biomassproduction. For example, plants that grow vegetatively have an increasebiomass production, compared to a plant of the same species that is notgenetically modified for substantial vegetative growth. Examples ofincreases in biomass production include increases of at least 5%, atleast 20%, or even at least 50%, when compared to an amount of biomassproduction by a plant of the same species not growing vegetatively.

The life cycle of flowering plants in general can be divided into threegrowth phases: vegetative, inflorescence, and floral (late inflorescencephase). In the vegetative phase, the shoot apical meristem (SAM)generates leaves that later will ensure the resources necessary toproduce fertile offspring. Upon receiving the appropriate environmentaland developmental signals the plant switches to floral, or reproductive,growth and the SAM enters the inflorescence phase (I) and gives rise toan inflorescence with flower primordia. During this phase the fate ofthe SAM and the secondary shoots that arise in the axils of the leavesis determined by a set of meristem identity genes, some of which preventand some of which promote the development of floral meristems. Onceestablished, the plant enters the late inflorescence phase where thefloral organs are produced. If the appropriate environmental anddevelopmental signals the plant switches to floral, or reproductive,growth are disrupted, the plant will not be able to enter reproductivegrowth, therefore maintaining vegetative growth.

Seed or seedling vigor is an important characteristic that can greatlyinfluence successful growth of a plant, such as crop plants. Adverseenvironmental conditions, such as dry, wet, cold or hot conditions, canaffect a plant growth cycle, and the vigor of seeds (i.e. vitality andstrength under such conditions can differentiate between successful andfailed crop growth). Seedling vigor has often been defined to comprisethe seed properties that determine “the potential for rapid, uniformemergence and development of normal seedlings under a wide range offield conditions”. Hence, it would be advantageous to develop plantseeds with increased vigor.

For example, increased seedling vigor would be advantageous for cerealplants such as rice, maize, wheat, etc. production. For these crops,growth can often be slowed or stopped by cool environmental temperaturesduring the planting season. In addition, rapid emergence and tilleringof rice would permit growers to initiate earlier flood irrigation whichcan save water and suppress weak growth. Genes associated with increasedseed vigor and/or cold tolerance in rice, have therefore been sought forproducing improve rice varieties. See e.g., Pinson, S., “MolecularMapping of Seedling Vigor QTLs in Tropical Rice”, USDA AgriculturalResearch Service, Dec. 16, 2000.

Seedling vigor has been measured by different tests and assays,including most typically a cold tolerance test and an accelerated agingtest.

Some of the nucleotide sequences of the invention code forbasic-helix-loop (bHCH) transcription factors. It is known thattranscription factors often control the expression of multiple genes ina pathway. The basic/helix-loop-helix (BHLH) proteins are a superfamilyof transcription factors that bind as dimers to specific DNA targetsites. The bHLH transcription factors have been well characterized innonplant eukaryotes and have been identified as important regulatorycomponents in diverse biological processes. Many different functionshave been identified for those proteins in animals, including thecontrol of cell proliferation and transcription often involves homo- orhetero-dimerization. Members of the RIB basic helix-loop-helix (bHLH)family of plant transcription factors are involved in a variety ofgrowth and differentiation processes.

A basic-helix-loop-helix (bHLH) is a protein structural motif thatcharacterizes a family of transcription factors. The motif ischaracterized by two α helices connected by a loop. Transcriptionfactors of this type are typically dimeric, each with one helixcontaining basic amino acid residues that facilitate DNA binding. Onehelix is typically smaller and due to the flexibility of the loop allowsdimerization by folding and packing against another helix. The largerhelix typically contains the DNA binding regions. bHLH proteinstypically bind to a consensus sequence called an E-box, CANNTG. Thecanonical E-box is CACGTG, however some bHLH transcription factors bindto different sequences, which are often similar to the E-box. bHLHtranscription factors are often important in development or cellactivity.

4. The Polypeptides/Polynucleotides of the Invention

The polynucleotides of the present invention and the proteins expressedvia translation of these polynucleotides are set forth in the SequenceListing, specifically SEQ ID NOS. 94, 95, 96, 97, 90, 91, 82, 83, 88,89, 84, 85, 86, 87, 92, 93, 80, 81. The Sequence Listing also consistsof functionally comparable proteins Polypeptides comprised of a sequencewithin and defined by one of the consensus sequences can be utilized forthe purposes of the invention, namely to make transgenic plants withmodulated biomass, growth rate and/or seedling vigor.

5. Use of the Polypeptides to make Transgenic Plants

To use the sequences of the present invention or a combination of themor parts and/or mutants and/or fusions and/or variants of them,recombinant DNA constructs are prepared that comprise the polynucleotidesequences of the invention inserted into a vector and that are suitablefor transformation of plant cells. The construct can be made usingstandard recombinant DNA techniques (see, Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, 1989, New York.) and can be introduced into the plantspecies of interest by, for example, Agrobacterium-mediatedtransformation, or by other means of transformation, for example, asdisclosed below.

The vector backbone may be any of those typically used in the field suchas plasmids, viruses, artificial chromosomes, BACs, YACs, PACs andvectors such as, for instance, bacteria-yeast shuttle vectors, lambdaphage vectors, T-DNA fusion vectors and plasmid vectors (see, Shizuya etal. (1992) Proc. Natl. Acad. Sci. USA, 89: 8794-8797; Hamilton et al.(1996) Proc. Natl. Acad. Sci. USA, 93: 9975-9979; Burke et al. (1987)Science, 236:806-812; Sternberg N. et al. (1990) Proc Natl Acad SciUSA., 87:103-7; Bradshaw et al. (1995) Nucl Acids Res, 23: 4850-4856;Frischauf et al. (1983) J. Mol Biol, 170: 827-842; Huynh et al., GloverN. Mex. (ed) DNA Cloning: A practical Approach, Vol. 1 Oxford: IRL Press(1985); Walden et al. (1990) Mol Cell Biol 1: 175-194).

Typically, the construct comprises a vector containing a nucleic acidmolecule of the present invention with any desired transcriptionaland/or translational regulatory sequences such as, for example,promoters, UTRs, and 3′ end termination sequences. Vectors may alsoinclude, for example, origins of replication, scaffold attachmentregions (SARs), markers, homologous sequences, and introns. The vectormay also comprise a marker gene that confers a selectable phenotype onplant cells. The marker may preferably encode a biocide resistancetrait, particularly antibiotic resistance, such as resistance to, forexample, kanamycin, bleomycin, or hygromycin, or herbicide resistance,such as resistance to, for example, glyphosate, chlorosulfuron orphosphinotricin.

It will be understood that more than one regulatory region may bepresent in a recombinant polynucleotide, e.g., introns, enhancers,upstream activation regions, transcription terminators, and inducibleelements. Thus, more than one regulatory region can be operably linkedto said sequence.

To “operably link” a promoter sequence to a sequence, the translationinitiation site of the translational reading frame of said sequence istypically positioned between one and about fifty nucleotides downstreamof the promoter. A promoter can, however, be positioned as much as about5,000 nucleotides upstream of the translation initiation site, or about2,000 nucleotides upstream of the transcription start site. A promotertypically comprises at least a core (basal) promoter. A promoter alsomay include at least one control element, such as an enhancer sequence,an upstream element or an upstream activation region (UAR). For example,a suitable enhancer is a cis-regulatory element (−212 to −154) from theupstream region of the octopine synthase (ocs) gene. Fromm et al., ThePlant Cell 1:977-984 (1989).

A basal promoter is the minimal sequence necessary for assembly of atranscription complex required for transcription initiation. Basalpromoters frequently include a “TATA box” element that may be locatedbetween about 15 and about 35 nucleotides upstream from the site oftranscription initiation. Basal promoters also may include a “CCAAT box”element (typically the sequence CCAAT) and/or a GGGCG sequence, whichcan be located between about 40 and about 200 nucleotides, typicallyabout 60 to about 120 nucleotides, upstream from the transcription startsite.

The choice of promoters to be included depends upon several factors,including, but not limited to, efficiency, selectability, inducibility,desired expression level, and cell- or tissue-preferential expression.It is a routine matter for one of skill in the art to modulate theexpression of a sequence by appropriately selecting and positioningpromoters and other regulatory regions relative to said sequence.

Some suitable promoters initiate transcription only, or predominantly,in certain cell types. For example, a promoter that is activepredominantly in a reproductive tissue (e.g., fruit, ovule, pollen,pistils, female gametophyte, egg cell, central cell, nucellus,suspensor, synergid cell, flowers, embryonic tissue, embryo sac, embryo,zygote, endosperm, integument, or seed coat) can be used. Thus, as usedherein a cell type- or tissue-preferential promoter is one that drivesexpression preferentially in the target tissue, but may also lead tosome expression in other cell types or tissues as well. Methods foridentifying and characterizing promoter regions in plant genomic DNAinclude, for example, those described in the following references:Jordano, et al., Plant Cell, 1:855-866 (1989); Bustos, et al., PlantCell, 1:839-854 (1989); Green, et al., EMBO J. 7, 4035-4044 (1988);Meier, et al., Plant Cell, 3, 309-316 (1991); and Zhang, et al., PlantPhysiology 110: 1069-1079 (1996).

Examples of various classes of promoters are described below. Some ofthe promoters indicated below are described in more detail in U.S.Patent Application Ser. Nos. 60/505,689; 60/518,075; 60/544,771;60/558,869; 60/583,691; 60/619,181; 60/637,140; Ser. Nos. 10/950,321;10/957,569; 11/058,689; 11/172,703; 11/208,308; and PCT/US05/23639. Itwill be appreciated that a promoter may meet criteria for oneclassification based on its activity in one plant species, and yet meetcriteria for a different classification based on its activity in anotherplant species.

Other Regulatory Regions: A 5′ untranslated region (UTR) can be includedin nucleic acid constructs described herein. A 5′ UTR is transcribed,but is not translated, and lies between the start site of the transcriptand the translation initiation codon and may include the +1 nucleotide.A 3′ UTR can be positioned between the translation termination codon andthe end of the transcript. UTRs can have particular functions such asincreasing mRNA stability or attenuating translation. Examples of 3′UTRs include, but are not limited to, polyadenylation signals andtranscription termination sequences, e.g., a nopaline synthasetermination sequence.

Various promoters can be used to drive expression of the polynucleotidesof the present invention. Nucleotide sequences of such promoters are setforth in SEQ ID NOS: 1-79. Some of them can be broadly expressingpromoters, others may be more tissue preferential.

A promoter can be said to be “broadly expressing” when it promotestranscription in many, but not necessarily all, plant tissues or plantcells. For example, a broadly expressing promoter can promotetranscription of an operably linked sequence in one or more of theshoot, shoot tip (apex), and leaves, but weakly or not at all in tissuessuch as roots or stems. As another example, a broadly expressingpromoter can promote transcription of an operably linked sequence in oneor more of the stem, shoot, shoot tip (apex), and leaves, but canpromote transcription weakly or not at all in tissues such asreproductive tissues of flowers and developing seeds. Non-limitingexamples of broadly expressing promoters that can be included in thenucleic acid constructs provided herein include the p326 (SEQ ID NO:76), YP0144 (SEQ ID NO: 55), YP0190 (SEQ ID NO: 59), p13879 (SEQ ID NO:75), YP0050 (SEQ ID NO: 35), p32449 (SEQ ID NO: 77), 21876 (SEQ ID NO:1), YP0158 (SEQ ID NO: 57), YP0214 (SEQ ID NO: 61), YP0380 (SEQ ID NO:70), PT0848 (SEQ ID NO: 26), and PT0633 (SEQ ID NO: 7). Additionalexamples include the cauliflower mosaic virus (CaMV) 35S promoter, themannopine synthase (MAS) promoter, the 1′ or 2′ promoters derived fromT-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34Spromoter, actin promoters such as the rice actin promoter, and ubiquitinpromoters such as the maize ubiquitin-1 promoter. In some cases, theCaMV 35S promoter is excluded from the category of broadly expressingpromoters.

Root-active promoters drive transcription in root tissue, e.g., rootendodermis, root epidermis, or root vascular tissues. In someembodiments, root-active promoters are root-preferential promoters,i.e., drive transcription only or predominantly in root tissue.Root-preferential promoters include the YP0128 (SEQ ID NO: 52), YP0275(SEQ ID NO: 63), PT0625 (SEQ ID NO: 6), PT0660 (SEQ ID NO: 9), PT0683(SEQ ID NO: 14), and PT0758 (SEQ ID NO: 22). Other root-preferentialpromoters include the PT0613 (SEQ ID NO: 5), PT0672 (SEQ ID NO: 11),PT0688 (SEQ ID NO: 15), and PT0837 (SEQ ID NO: 24), which drivetranscription primarily in root tissue and to a lesser extent in ovulesand/or seeds. Other examples of root-preferential promoters include theroot-specific subdomains of the CaMV 35S promoter (Lam et al., Proc.Natl. Acad. Sci. USA 86:7890-7894 (1989)), root cell specific promotersreported by Conkling et al., Plant Physiol. 93:1203-1211 (1990), and thetobacco RD2 gene promoter.

In some embodiments, promoters that drive transcription in maturingendosperm can be useful. Transcription from a maturing endospermpromoter typically begins after fertilization and occurs primarily inendosperm tissue during seed development and is typically highest duringthe cellularization phase. Most suitable are promoters that are activepredominantly in maturing endosperm, although promoters that are alsoactive in other tissues can sometimes be used. Non-limiting examples ofmaturing endosperm promoters that can be included in the nucleic acidconstructs provided herein include the napin promoter, the Arcelin-5promoter, the phaseolin gene promoter (Bustos et al. (1989) Plant Cell1(9):839-853), the soybean trypsin inhibitor promoter (Riggs et al.(1989) Plant Cell 1(6):609-621), the ACP promoter (Baerson et al. (1993)Plant Mol Biol, 22(2):255-267), the stearoyl-ACP desaturase gene(Slocombe et al. (1994) Plant Physiol 104(4):167-176), the soybean α′subunit of β-conglycinin promoter (Chen et al. (1986) Proc Natl Acad SciUSA 83:8560-8564), the oleosin promoter (Hong et al. (1997) Plant MolBiol 34(3):549-555), and zein promoters, such as the 15 kD zeinpromoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kD zeinpromoter and 27 kD zein promoter. Also suitable are the Osgt-1 promoterfrom the rice glutelin-1 gene (Zheng et al. (1993) Mol. Cell Biol.13:5829-5842), the beta-amylase gene promoter, and the barley hordeingene promoter. Other maturing endosperm promoters include the YP0092(SEQ ID NO: 38), PT0676 (SEQ ID NO: 12), and PT0708 (SEQ ID NO: 17.

Promoters that drive transcription in ovary tissues such as the ovulewall and mesocarp can also be useful, e.g., a polygalacturonidasepromoter, the banana TRX promoter, and the melon actin promoter. Othersuch promoters that drive gene expression preferentially in ovules areYP0007 (SEQ ID NO: 30), YP011 (SEQ ID NO: 46), YP0092 (SEQ ID NO: 38),YP0103 (SEQ ID NO: 43), YP0028 (SEQ ID NO: 33), YP0121 (SEQ ID NO: 51),YP0008 (SEQ ID NO: 31), YP0039 (SEQ ID NO: 34), YP0115 (SEQ ID NO: 47),YP0119 (SEQ ID NO: 49), YP0120 (SEQ ID NO: 50) and YP0374 (SEQ ID NO:68).

In some other embodiments of the present invention, embryo sac/earlyendosperm promoters can be used in order drive transcription of thesequence of interest in polar nuclei and/or the central cell, or inprecursors to polar nuclei, but not in egg cells or precursors to eggcells. Most suitable are promoters that drive expression only orpredominantly in polar nuclei or precursors thereto and/or the centralcell. A pattern of transcription that extends from polar nuclei intoearly endospern development can also be found with embryo sac/earlyendosperm-preferential promoters, although transcription typicallydecreases significantly in later endosperm development during and afterthe cellularization phase. Expression in the zygote or developing embryotypically is not present with embryo sac/early endosperm promoters.

Promoters that may be suitable include those derived from the followinggenes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsisatmycl (see, Urao (1996) Plant Mol. Biol., 32:571-57; Conceicao (1994)Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); ArabidopsisMEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Pat. No.6,906,244). Other promoters that may be suitable include those derivedfrom the following genes: maize MAC1 (see, Sheridan (1996) Genetics,142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) PlantMol. Biol., 22:10131-1038). Other promoters include the followingArabidopsis promoters: YP0039 (SEQ ID NO: 34), YP0101 (SEQ ID NO: 41),YP0102 (SEQ ID NO: 42), YP0110 (SEQ ID NO: 45), YP0117 (SEQ ID NO: 48),YP0119 (SEQ ID NO: 49), YP0137 (SEQ ID NO: 53), DME, YP0285 (SEQ ID NO:64), and YP0212 (SEQ ID NO: 60). Other promoters that may be usefulinclude the following rice promoters: p530c10, pOsFIE2-2, pOsMEA,pOsYp102, and pOsYp285.

Promoters that preferentially drive transcription in zygotic cellsfollowing fertilization can provide embryo-preferential expression andmay be useful for the present invention. Most suitable are promotersthat preferentially drive transcription in early stage embryos prior tothe heart stage, but expression in late stage and maturing embryos isalso suitable. Embryo-preferential promoters include the barley lipidtransfer protein (Ltp1) promoter (Plant Cell Rep (2001) 20:647-654,YP0097 (SEQ ID NO: 40), YP0107 (SEQ ID NO: 44), YP0088 (SEQ ID NO: 37),YP0143 (SEQ ID NO: 54), YP0156 (SEQ ID NO: 56), PT0650 (SEQ ID NO: 8),PT0695 (SEQ ID NO: 16), PT0723 (SEQ ID NO: 19), PT0838 (SEQ ID NO: 25),PT0879 (SEQ ID NO: 28) and PT0740 (SEQ ID NO: 20).

Promoters active in photosynthetic tissue in order to drivetranscription in green tissues such as leaves and stems are ofparticular interest for the present invention. Most suitable arepromoters that drive expression only or predominantly such tissues.Examples of such promoters include the ribulose-1,5-bisphosphatecarboxylase (RbcS) promoters such as the RbcS promoter from easternlarch (Larix laricina), the pine cab6 promoter (Yamamoto et al. (1994)Plant Cell Physiol. 35:773-778), the Cab-1 gene promoter from wheat(Fejes et al. (1990) Plant Mol. Biol. 15:921-932), the CAB-1 promoterfrom spinach (Lubberstedt et al. (1994) Plant Physiol. 104:997-1006),the cab1R promoter from rice (Luan et al. (1992) Plant Cell 4:971-981),the pyruvate orthophosphate dikinase (PPDK) promoter from corn (Matsuokaet al. (1993) Proc Natl Acad. Sci USA 90:9586-9590), the tobacco Lhcb1*2promoter (Cerdan et al. (1997) Plant Mol. Biol. 33:245-255), theArabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit et al.(1995) Planta 196:564-570), and thylakoid membrane protein promotersfrom spinach (psad, psaF, psae, PC, FNR, atpC, atpD, cab, rbcS. Otherpromoters that drive transcription in stems, leafs and green tissue arePT0535 (SEQ ID NO: 3), PT0668 (SEQ ID NO: 2), PT0886 (SEQ ID NO: 29),PR0924 (SEQ ID NO: 78), YP0144 (SEQ ID NO: 55), YP0380 (SEQ ID NO: 70)and PT0585 (SEQ ID NO: 4).

In some other embodiments of the present invention, inducible promotersmay be desired. Inducible promoters drive transcription in response toexternal stimuli such as chemical agents or environmental stimuli. Forexample, inducible promoters can confer transcription in response tohormones such as giberellic acid or ethylene, or in response to light ordrought. Examples of drought inedible promoters are YP0380 (SEQ ID NO:70), PT0848 (SEQ ID NO: 26), YP0381 (SEQ ID NO: 71), YP0337 (SEQ ID NO:66), YP0337 (SEQ ID NO: 66), PT0633 (SEQ ID NO: 7), YP0374 (SEQ ID NO:68), PT0710 (SEQ ID NO: 18), YP0356 (SEQ ID NO: 67), YP0385 (SEQ ID NO:73), YP0396 (SEQ ID NO: 74), YP0384 (SEQ ID NO: 72), YP0384 (SEQ ID NO:72), PT0688 (SEQ ID NO: 15), YP0286 (SEQ ID NO: 65), YP0377 (SEQ ID NO:69), and PD1367 (SEQ ID NO: 79). Examples of promoters induced bynitrogen are PT0863 (SEQ ID NO: 27), PT0829 (SEQ ID NO: 23), PT0665 (SEQID NO: 10) and PT0886 (SEQ ID NO: 29). An example of a shade induciblepromoter is PR0924 (SEQ ID NO: 78).

Other Promoters: Other classes of promoters include, but are not limitedto, leaf-preferential, stem/shoot-preferential, callus-preferential,guard cell-preferential, such as PT0678 (SEQ ID NO: 13), andsenescence-preferential promoters. Promoters designated YP0086 (SEQ IDNO: 36), YP0188 (SEQ ID NO: 58), YP0263 (SEQ ID NO: 62), PT0758 (SEQ IDNO: 22), PT0743 (SEQ ID NO: 21), PT0829 (SEQ ID NO: 23), YP0119 (SEQ IDNO: 49), and YP0096 (SEQ ID NO: 39), as described in theabove-referenced patent applications, may also be useful.

Alternatively, misexpression can be accomplished using a two componentsystem, whereby the first component consists of a transgenic plantcomprising a transcriptional activator operatively linked to a promoterand the second component consists of a transgenic plant that comprise anucleic acid molecule of the invention operatively linked to thetarget-binding sequence/region of the transcriptional activator. The twotransgenic plants are crossed and the nucleic acid molecule of theinvention is expressed in the progeny of the plant. In anotheralternative embodiment of the present invention, the misexpression canbe accomplished by having the sequences of the two component systemtransformed in one transgenic plant line.

Another alternative consists in inhibiting expression of a biomass orvigor-modulating polypeptide in a plant species of interest. The term“expression” refers to the process of converting genetic informationencoded in a polynucleotide into RNA through transcription of thepolynucleotide (i.e., via the enzymatic action of an RNA polymerase),and into protein, through translation of mRNA. “Up-regulation” or“activation” refers to regulation that increases the production ofexpression products relative to basal or native states, while“down-regulation” or “repression” refers to regulation that decreasesproduction relative to basal or native states.

A number of nucleic-acid based methods, including anti-sense RNA,ribozyme directed RNA cleavage, and interfering RNA (RNAi) can be usedto inhibit protein expression in plants. Antisense technology is onewell-known method. In this method, a nucleic acid segment from theendogenous gene is cloned and operably linked to a promoter so that theantisense strand of RNA is transcribed. The recombinant vector is thentransformed into plants, as described above, and the antisense strand ofRNA is produced. The nucleic acid segment need not be the entiresequence of the endogenous gene to be repressed, but typically will besubstantially identical to at least a portion of the endogenous gene tobe repressed. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Typically, a sequence of at least 30nucleotides is used (e.g., at least 40, 50, 80, 100, 200, 500nucleotides or more).

Thus, for example, an isolated nucleic acid provided herein can be anantisense nucleic acid to one of the aforementioned nucleic acidsencoding a biomass-modulating polypeptide. A nucleic acid that decreasesthe level of a transcription or translation product of a gene encoding abiomass-modulating polypeptide is transcribed into an antisense nucleicacid similar or identical to the sense coding sequence of the biomass-or growth rate-modulating polypeptide. Alternatively, the transcriptionproduct of an isolated nucleic acid can be similar or identical to thesense coding sequence of a biomass growth rate-modulating polypeptide,but is an RNA that is unpolyadenylated, lacks a 5′ cap structure, orcontains an unsplicable intron.

In another method, a nucleic acid can be transcribed into a ribozylne,or catalytic RNA, that affects expression of an mRNA. (See, U.S. Pat.No. 6,423,885). Ribozymes can be designed to specifically pair withvirtually any target RNA and cleave the phosphodiester backbone at aspecific location, thereby functionally inactivating the target RNA.Heterologous nucleic acids can encode ribozymes designed to cleaveparticular mRNA transcripts, thus preventing expression of apolypeptide. Hammerhead ribozymes are useful for destroying particularmRNAs, although various ribozymes that cleave mRNA at site-specificrecognition sequences can be used. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target RNAcontain a 5′-UG-3′ nucleotide sequence. The construction and productionof hammerhead ribozymes is known in the art. See, for example, U.S. Pat.No. 5,254,678 and WO 02/46449 and references cited therein. Hammerheadribozyme sequences can be embedded in a stable RNA such as a transferRNA (tRNA) to increase cleavage efficiency in vivo. Perriman, et al.(1995) Proc. Natl. Acad. Sci. USA, 92(13):6175-6179; de Feyter andGaudron, Methods in Molecular Biology, Vol. 74, Chapter 43, “ExpressingRibozymes in Plants”, Edited by Turner, P. C, Humana Press Inc., Totowa,N.J. RNA endoribonucleases such as the one that occurs naturally inTetrahymena thermophila, and which have been described extensively byCech and collaborators can be useful. See, for example, U.S. Pat. No.4,987,071.

Methods based on RNA interference (RNAi) can be used. RNA interferenceis a cellular mechanism to regulate the expression of genes and thereplication of viruses. This mechanism is thought to be mediated bydouble-stranded small interfering RNA molecules. A cell responds to sucha double-stranded RNA by destroying endogenous mRNA having the samesequence as the double-stranded RNA. Methods for designing and preparinginterfering RNAs are known to those of skill in the art; see, e.g., WO99/32619 and WO 01/75164. For example, a construct can be prepared thatincludes a sequence that is transcribed into an interfering RNA. Such anRNA can be one that can anneal to itself, e.g., a double stranded RNAhaving a stem-loop structure. One strand of the stem portion of a doublestranded RNA comprises a sequence that is similar or identical to thesense coding sequence of the polypeptide of interest, and that is fromabout 10 nucleotides to about 2,500 nucleotides in length. The length ofthe sequence that is similar or identical to the sense coding sequencecan be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25nucleotides to 100 nucleotides. The other strand of the stem portion ofa double stranded RNA comprises an antisense sequence of thebiomass-modulating polypeptide of interest, and can have a length thatis shorter, the same as, or longer than the corresponding length of thesense sequence. The loop portion of a double stranded RNA can be from 10nucleotides to 5,000 nucleotides, e.g., from 15 nucleotides to 1,000nucleotides, from 20 nucleotides to 500 nucleotides, or from 25nucleotides to 200 nucleotides. The loop portion of the RNA can includean intron. See, e.g., WO 99/53050.

In some nucleic-acid based methods for inhibition of gene expression inplants, a suitable nucleic acid can be a nucleic acid analog. Nucleicacid analogs can be modified at the base moiety, sugar moiety, orphosphate backbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six-membered morpholino ring, or peptidenucleic acids, in which the deoxyphosphate backbone is replaced by apseudopeptide backbone and the four bases are retained. See, forexample, Summerton and Weller, 1997, Antisense Nucleic Acid Drug Dev.,7:187-195; Hyrup et al., 1996, Bioorgan. Med. Chem., 4: 5-23. Inaddition, the deoxyphosphate backbone can be replaced with, for example,a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite,or an alkyl phosphotriester backbone.

Transformation

Nucleic acid molecules of the present invention may be introduced intothe genome or the cell of the appropriate host plant by a variety oftechniques. These techniques, able to transform a wide variety of higherplant species, are well known and described in the technical andscientific literature (see, e.g., Weising et al. (1988) Ann. Rev.Genet., 22:421 and Christou (1995) Euphytica, 85:13-27).

A variety of techniques known in the art are available for theintroduction of DNA into a plant host cell. These techniques includetransformation of plant cells by injection (Newell (2000)),microinjection (Griesbach (1987) Plant Sci. 50:69-77), electroporationof DNA (Fromm et al. (1985) Proc. Natl. Acad. Sci. USA 82:5824), PEG(Paszkowski et al. (1984) EMBO J. 3:2717), use of biolistics (Klein etal. (1987) Nature 327:773), fusion of cells or protoplasts (Willmitzer,L. (1993) Transgenic Plants. In: Iotechnology, A Multi-VolumeComprehensive treatise (H. J. Rehm, G. Reed, A. Püler, P. Stadler, eds.,Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge), and via T-DNAusing Agrobacterium tumefaciens (Crit. Rev. Plant. Sci. 4:1-46; Fromm etal. (1990) Biotechnology 8:833-844) or Agrobacterium rhizogenes (Cho etal. (2000) Planta 210:195-204) or other bacterial hosts (Brootghaerts etal. (2005) Nature 433:629-633), for example.

In addition, a number of non-stable transformation methods that are wellknown to those skilled in the art may be desirable for the presentinvention. Such methods include, but are not limited to, transientexpression (Lincoln et al. (1998) Plant Mol. Biol. Rep. 16:1-4) andviral transfection (Lacomme et al. (2001), “Genetically EngineeredViruses” (C. J. A. Ring and E. D. Blair, Eds). Pp. 59-99, BIOSScientific Publishers, Ltd. Oxford, UK).

Seeds are obtained from the transformed plants and used for testingstability and inheritance. Generally, two or more generations arecultivated to ensure that the phenotypic feature is stably maintainedand transmitted.

A person of ordinary skill in the art recognizes that after theexpression cassette is stably incorporated in transgenic plants andconfirmed to be operable, it can be introduced into other plants bysexual crossing. Any of a number of standard breeding techniques can beused, depending upon the species to be crossed.

The nucleic acid molecules of the present invention may be used toconfer the trait of an altered flowering time.

The nucleic acid molecules of the present invention encode appropriateproteins from any organism, but are preferably found in plants, fungi,bacteria or animals.

The methods according to the present invention can be applied to anyplant, preferably higher plants, pertaining to the classes ofAngiospermae and Gymnospermae. Plants of the subclasses of theDicotylodenae and the Monocotyledonae are particularly suitable.Dicotyledonous plants belonging to the orders of the Magniolales,illiciales, Laurales, Piperales Aristochiales, Nymphaeales,Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales,Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales,Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales,Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violales,Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales,Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales,Proteales, Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales,Sapindales, Juglandales, Geraniales, Polygalales, Umbellales,Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales,Campanulales, Rubiales, Dipsacales, and Asterales, for example, are alsosuitable. Monocotyledonous plants belonging to the orders of theAlismatales, Hydrocharitales, Najadales, Triuridales, Commelinales,Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales,Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales,Lilliales, and Orchidales also may be useful in embodiments of thepresent invention. Further examples include, but are not limited to,plants belonging to the class of the Gymnospermae are Pinales,Ginkgoales, Cycadales and Gnetales.

The methods of the present invention are preferably used in plants thatare important or interesting for agriculture, horticulture, biomass forbioconversion and/or forestry. Non-limiting examples include, forinstance, tobacco, oilseed rape, sugar beet, potatoes, tomatoes,cucumbers, peppers, beans, peas, citrus fruits, avocados, peaches,apples, pears, berries, plumbs, melons, eggplants, cotton, soybean,sunflowers, roses, poinsettia, petunia, guayule, cabbages, spinach,alfalfa, artichokes, sugarcane, mimosa, Servicea lespedera, corn, wheat,rice, rye, barley, sorghum and grasses such as switch grass, giant reed,Bermuda grass, Johnson grasses or turf grass, millet, hemp, bananas,poplars, eucalyptus trees and conifers. Of interest are plates grown forenergy production, so called energy crops, such as broadleaf plants likealfalfa, hemp, Jerusalem artichoke and grasses such as sorgum,switchgrass, Johnson grass and the likes.

Homologues Encompassed by the Invention

It is known in the art that one or more amino acids in a sequence can besubstituted with other amino acid(s), the charge and polarity of whichare similar to that of the substituted amino acid, i.e. a conservativeamino acid substitution, resulting in a biologically/functionally silentchange. Conservative substitutes for an amino acid within thepolypeptide sequence can be selected from other members of the class towhich the amino acid belongs. Amino acids can be divided into thefollowing four groups: (1) acidic (negatively charged) amino acids, suchas aspartic acid and glutamic acid; (2) basic (positively charged) aminoacids, such as arginine, histidine, and lysine; (3) neutral polar aminoacids, such as serine, threonine, tyrosine, asparagine, and glutamine;and (4) neutral nonpolar (hydrophobic) amino acids such as glycine,alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, cysteine, and methionine.

Nucleic acid molecules of the present invention can comprise sequencesthat differ from those encoding a protein or fragment thereof selectedfrom the group consisting of Leads 80, 81, 113, 114, ME08328, ME01905,ME01770, ME21445 and ME20023, SEQ ID NOS. 95, 97, 91, 83, 89, 85, 87,93, and 81, respectively, due to the fact that the different nucleicacid sequence encodes a protein having one or more conservative aminoacid changes.

Biologically functional equivalents of the polypeptides, or fragmentsthereof, of the present invention can have about 10 or fewerconservative amino acid changes, more preferably about 7 or fewerconservative amino acid changes, and most preferably about 5 or fewerconservative amino acid changes. In a preferred embodiment of thepresent invention, the polypeptide has between about 5 and about 500conservative changes, more preferably between about 10 and about 300conservative changes, even more preferably between about 25 and about150 conservative changes, and most preferably between about 5 and about25 conservative changes or between 1 and about 5 conservative changes.

Identification of Useful Nucleic Acid Molecules and Their CorrespondingNucleotide Sequences

The nucleic acid molecules, and nucleotide sequences thereof, of thepresent invention were identified by use of a variety of screens thatare predictive of nucleotide sequences that provide plants with alteredsize, vegetative growth, growth rate, organ number, plant architectureand/or biomass. One or more of the following screens were, therefore,utilized to identify the nucleotide (and amino acid) sequences of thepresent invention.

The present invention is further exemplified by the following examples.The examples are not intended to in any way limit the scope of thepresent application and its uses.

6. Experiments Confirming the Usefulness of the Polynucleotides andPolypeptides of the Invention

General Protocols

Agrobacterium-Mediated Transformation of Arabidopsis

Wild-type Arabidopsis thaliana Wassilewskija (WS) plants are transformedwith Ti plasmids containing clones in the sense orientation relative tothe 35S promoter. A Ti plasmid vector useful for these constructs, CRS338, contains the Ceres-constructed, plant selectable marker genephosphinothricin acetyltransferase (PAT), which confers herbicideresistance to transformed plants.

Ten independently transformed events are typically selected andevaluated for their qualitative phenotype in the T₁ generation.

Preparation of Soil Mixture: 24 L SunshineMix #5 soil (Sun GroHorticulture, Ltd., Bellevue, Wash.) is mixed with 16 L Therm-O-Rockvermiculite (Therm-O-Rock West, Inc., Chandler, Ariz.) in a cement mixerto make a 60:40 soil mixture. To the soil mixture is added 2 TbspMarathon 1% granules (Hummert, Earth City, Mo.), 3 Tbsp OSMOCOTE®14-14-14 (Hummert, Earth City, Mo.) and 1 Tbsp Peters fertilizer20-20-20 (J. R. Peters, Inc., Allentown, Pa.), which are first added to3 gallons of water and then added to the soil and mixed thoroughly.Generally, 4-inch diameter pots are filled with soil mixture. Pots arethen covered with 8-inch squares of nylon netting.

Planting: Using a 60 mL syringe, 35 mL of the seed mixture is aspirated.25 drops are added to each pot. Clear propagation domes are placed ontop of the pots that are then placed under 55% shade cloth andsubirrigated by adding 1 inch of water.

Plant Maintenance: 3 to 4 days after planting, lids and shade cloth areremoved. Plants are watered as needed. After 7-10 days, pots are thinnedto 20 plants per pot using forceps. After 2 weeks, all plants aresubirrigated with Peters fertilizer at a rate of 1 Tsp per gallon ofwater. When bolts are about 5-10 cm long, they are clipped between thefirst node and the base of stem to induce secondary bolts. Dippinginfiltration is performed 6 to 7 days after clipping.

Preparation of Agrobacterium: To 150 mL fresh YEB is added 0.1 mL eachof carbenicillin, spectinomycin and rifampicin (each at 100 mg/ml stockconcentration). Agrobacterium starter blocks are obtained (96-well blockwith Agrobacterium cultures grown to an OD₆₀₀ of approximately 1.0) andinoculated one culture vessel per construct by transferring 1 mL fromappropriate well in the starter block. Cultures are then incubated withshaking at 27° C. Cultures are spun down after attaining an OD₆₀₀ ofapproximately 1.0 (about 24 hours). 200 mL infiltration media is addedto resuspend Agrobacterium pellets. Infiltration media is prepared byadding 2.2 g MS salts, 50 g sucrose, and 5±2 mg/ml benzylaminopurine to900 ml water.

Dipping Infiltration: The pots are inverted and submerged for 5 minutesso that the aerial portion of the plant is in the Agrobacteriumsuspension. Plants are allowed to grow normally and seed is collected.

High-throughput Phenotypic Screening of Misexpression Mutants: Seed isevenly dispersed into water-saturated soil in pots and placed into adark 4° C. cooler for two nights to promote uniform germination. Potsare then removed from the cooler and covered with 55% shade cloth for4-5 days. Cotyledons are fully expanded at this stage. FINALE® (SanofiAventis, Paris, France) is sprayed on plants (3 ml FINALE® diluted into48 oz. water) and repeated every 3-4 days until only transformantsremain. Screening: Screening is routinely performed at four stages:Seedling, Rosette, Flowering, and Senescence.

-   -   Seedling—the time after the cotyledons have emerged, but before        the 3^(rd) true leaf begins to form.    -   Rosette—the time from the emergence of the 3^(rd) true leaf        through just before the primary bolt begins to elongate.    -   Flowering—the time from the emergence of the primary bolt to the        onset of senescence (with the exception of noting the flowering        time itself, most observations should be made at the stage where        approximately 50% of the flowers have opened).    -   Senescence—the time following the onset of senescence (with the        exception of “delayed senescence”, most observations should be        made after the plant has completely dried). Seeds are then        collected.

Screens: Screening for increased size, vegetative growth and/or biomassis performed by taking measurements, specifically T₂ measurements weretaken as follows:

-   -   Days to Bolt=number of days between sowing of seed and emergence        of first inflorescence.    -   Rosette Leaf Number at Bolt=number of rosette leaves present at        time of emergence of first inflorescence.    -   Rosette Area=area of rosette at time of initial inflorescence        emergence, using formula ((L×W)*3.14)/4.    -   Height=length of longest inflorescence from base to apex. This        measurement was taken at the termination of flowering/onset of        senescence.    -   Primary Inflorescence Thickness=diameter of primary        inflorescence 2.5 cm up from base. This measurement was taken at        the termination of flowering/onset of senescence.    -   Inflorescence Number=total number of unique inflorescences. This        measurement was taken at the termination of flowering/onset of        senescence.

PCR was used to amplify the cDNA insert in one randomly chosen T₂ plant.This PCR product was then sequenced to confirm the sequence in theplants.

Screening Superpools For Tolerance To Low Ammonium Nitrate GrowthConditions:

Superpools are generated and two thousand seeds each from ten superpoolsare pooled together and assayed using the Low Ammonium Nitrate Screen onAgar. Low ammonium nitrate growth media, pH 5.7, is as follows: 0.5×MSwithout N (PhytoTech), 0.5% sucrose (Sigma), 240 μM NH₄NO₃ (EM Science),0.5 g MES hydrate (Sigma), 0.8% Phytagar (EM Science). Forty-five (45)ml of media per square plate is used.

Arabidopsis thaliana cv WS seed is sterilized in 50% Clorox™ with 0.01%Triton X-100 (v/v) for five minutes, washed four times with steriledistilled deionized water and stored at 4° C. in the dark for 3 daysprior to use.

Seed is plated at a density of 100 seeds per plate. Wild-type seed isused as a control. Plates are incubated in a Conviron™ growth chamber at22° C. with a 16:8 hour light:dark cycle from a combination ofincandescent and fluorescent lamps emitting a light intensity of ˜100μEeinsteins and 70% humidity.

Seedlings are screened daily after 14 days. Candidate seedlings arelarger or stay greener longer relative to wild-type controls. DNA isisolated from each candidate plant and sequenced to determine whichtransgene was present.

Seedling Low Ammonium Nitrate Assay On Agar:

Media and seeds are prepared as described above.

Seeds from five misexpression line events, each containing the samepolynucleotide, are sown in two rows, with ten seeds per row. Each platecontains five events, for a total of 100 seeds. Control platescontaining wild-type seed are also prepared. Plates are then incubatedat 4° C. for at least two days.

After the several day 4° C. cold treatment, plates are incubated in aConviron™ growth chamber at 22° C. with a 16:8 hour light: dark cyclefrom a combination of incandescent and fluorescent lamps emitting alight intensity of ˜100 [Einsteins and 70% humidity.

After 14 days, plates are scanned daily using a CF Imager (TechnologicaLtd.) with a 45 minute dark acclimation. The CF Imager is used toquantify the seedlings' optimum quantum yields (Fv/Fm) as a measure ofphotosynthetic health (see details below). To quantify the seedlings'sizes, plates are also scanned with a flatbed photo scanner (Epson) oneday after nitrogen stress is apparent and wild-type seedling growth isarrested. Image capture is ended after all wild-type plants havecompletely yellowed. On the final scanning day plates are uncovered andliberally sprayed with Finale™ (10 ml in 48 oz. Murashige & Skoog liquidmedia) and returned to the growth chamber.

Two days after spraying, the plates are placed in a closed box for 45minutes to acclimate in preparation for fluorescence visualization viaCF Imager. Plants resistant to Finale™ appear red while sensitive plantsappear blue. After image capture, plants are assigned a transgenic(resistant) or non-transgenic (sensitive) status. The non-transgenicplants (i.e. non-transgenic segregants) serve as internal controls.

Seedling photosynthetic efficiency, or electron transport viaphotosystem II, is estimated by the relationship between Fm, the maximumfluorescence signal and the variable fluorescence, Fv. Here, a reductionin the optimum quantum yield (Fv/Fm) indicates stress, and so can beused to monitor the performance of transgenic plants compared tonon-transgenic plants under nitrogen stress conditions. Since a largeamount of nitrogen is invested in maintaining the photosyntheticapparatus, nitrogen deficiencies can lead to dismantling of the reactioncenters and to reductions in photosynthetic efficiency. Consequently,from the start of image capture collection until the plants are dead theFv/Fm ratio is determined for each seedling using the FluroImager 2software (Kevin Oxborough and John Bartington).

The rosette area of each plant is also analyzed using WinRHIZO software(Regent Instruments) to analyze the Epson flatbed scanner capturedimages.

Low Ammonium Nitrate Validated Assay:

Media and seeds are prepared as described above.

For misexpression lines which pass the above low ammonium nitrate assay,both T₂ and T₃ generation seed for an event are plated along withwild-type seed, at a final density of 100 seeds per plate. Platescontain 10 seed/row and have four rows of 10 T₂ seed followed by tworows of wild-type seed, followed by four rows of T₃ seed. Plates arethen incubated at 4° C. for at least two days.

After the several day 4° C. cold treatment, plates are incubated in aConviron™ growth chamber at 22° C. with a 16:8 hour light:dark cyclefrom a combination of incandescent and fluorescent lamps emitting alight intensity of ˜100 μEinsteins and 70% humidity.

After 14 days, plates are scanned daily using a CF Imager (TechnologicaLtd.) with a 45 minute dark acclimation. The CF Imager is used toquantify the seedlings' optimum quantum yields (Fv/Fm) as a measure ofphotosynthetic health. To quantify the seedlings' sizes, plates are alsoscanned with a flatbed photo scanner (Epson) one day after nitrogenstress is apparent and wild-type seedling growth is arrested. Imagecapture is ended after all wild-type plants have completely yellowed. Onthe final scanning day plates are uncovered and liberally sprayed withFinale™ (10 ml in 48 oz. Murashige & Skoog liquid media) and returned tothe growth chamber.

Two days after spraying, the plates are placed in a closed box for 45minutes to acclimate in preparation for fluorescence visualization viaCF Imager. Plants resistant to Finale™ appear red while sensitive plantsappear blue. After image capture, plants are assigned a transgenic(resistant) or non-transgenic (sensitive) status. The non-transgenicplants (i.e. non-transgenic segregants) serve as internal controls.

Fv/Fm ratio is determined for each seedling using the FluroImager 2software (Kevin Oxborough and John Bartington).

The rosette area of each plant is also analyzed using WinRHIZO software(Regent Instruments) to analyze the Epson flatbed scanner capturedimages.

Results:

Plants transformed with the genes of interest were screened as describedabove for modulated growth and phenotype characteristics. Theobservations include those with respect to the entire plant, as well asparts of the plant, such as the roots and leaves. The observations fortransformants with each polynucleotide sequence are noted in theSequence listing for each of the tested nucleotide sequences and thecorresponding encoded polypeptide. The modulated characteristics (i.e.observed phenotypes) are noted by an entry in the “miscellaneousfeatures” field for each respective sequence. The “Phenotype” noted inthe Sequence Listing for each relevant sequence further includes astatement of the useful utility of that sequence based on theobservations.

The observations made for the various transformants can be categorized,depending upon the relevant plant tissue for the observation and theconsequent utility/usefulness of the nucleotide sequence/polypeptideused to make that transformant. Table 1 correlates the shorthand notesin the sequence listing to the observations noted for each tranformant(the “description” column), the tissue of the observation, the phenotypethereby associated with the transformant, and the consequentutility/usefulness of the inserted nucleotide sequence and encodedpolypeptide (the “translation” column).

For some of the polynucleotides/polypeptides of the invention, thesequence listing further includes (in a “miscellaneous feature” section)an indication of important identified dominant(s) and the correspondingfunction of the domain or identified by comparison to the publiclyavailable pfam database. TABLE 1 PHENOTYPE TISSUE QUALIFIER PHENOTYPEDESCRIPTION TRANSLATION WHOLE Senescence Time Early the plant senescesUseful for accelerating PLANT Senescence significantly early cropdevelopment and (note the approximate harvest number of days early itstarted to senesce in the comments) INFLORESCENCE Flowering Time EarlyFlowering the plant flowers Useful for accelerating significantly earlyflowering time (note the approximate number of days early it flowered inthe comments) INFLORESCENCE Flowering Time Late Flowering the plantflowers Useful for delaying significantly late flowering time (note theapproximate number of days late it flowered in the comments)INFLORESCENCE Flowering Time Dtb days to bolt Useful for delayingflowering time WHOLE Senescence Time Late Senescence the plant senescesUseful for delaying PLANT significantly late senescence (note theapproximate number of days late it started to senesce in the comments)COTYLEDONS Silver Silver cotyledons have a Useful for drought orgray/silver colored stress tolerance surface; This phenotype is oftenaccompanied by a small size mutation, but not always WHOLE Dark GreenDark Green plant is visibly darker Useful for increasing SEEDLING greenchlorophyll and photosynthetic capacity WHOLE Color Dark Green the plantis Useful for increasing PLANT abnormally dark chlorophyll and greenphotosynthetic capacity WHOLE High High the plant is purple in Usefulfor increasing SEEDLING Anthocyanin Anthocyanin color increasinganthocyanin content WHOLE Color High the plant is purple in Useful forincreasing PLANT Anthocyanin color increasing anthocyanin content ROOTNo Growth in No Growth in roots grow along the Useful for increasingroot Soil Soil soil surface instead of growth eg to enhance into thesoil nutrient uptake ROOT Other Other this correlates with Useful forincreasing root any root mutant growth eg to enhance phenotypes which donutrient uptake not fit into the above categories (a picture should betaken for documentation) LATERAL Number Less Lateral there is an Usefulfor increasing root ROOTS Roots abnormally low growth eg to enhancenumber of lateral nutrient uptake roots LATERAL Other Other thiscorrelates with Useful for increasing root ROOTS any lateral root growtheg to enhance mutant phenotypes nutrient uptake which do not fit intothe above categories (a picture should be taken for documentation) ROOTClassic Classic there is a lack of Useful for increasing root lateralroots (buds growth eg to enhance may appear but do nutrient uptake notelongate) ROOT Dwarf Dwarf there is a stunted root Useful for increasingroot system growth eg to enhance nutrient uptake ROOT Mid-SectionMid-Section there are lateral roots Useful for increasing root in thetop and bottom growth eg to enhance quarters of the whole nutrientuptake root, but none in the middle ROOT Split Split appears as“classic” Useful for increasing root but with two primary growth eg toenhance roots, both nutrient uptake originating from the hypocotyl baseROOT Other Other this correlates with Useful for increasing root anyoverall root growth eg to enhance structure mutant nutrient uptakephenotypes which do not fit into the above categories (a picture shouldbe taken for documentation) PRIMARY Other Other this correlates withUseful for increasing root ROOT any primary root growth eg to enhancemutant phenotypes nutrient uptake which do not fit into the abovecategories (a picture should be taken for documentation) ROOT LengthLonger Root the root hairs are Useful for increasing root HAIRS Hairabnormally long growth eg to enhance nutrient uptake ROOT Length SmallerRoot the root hairs are Useful for increasing root HAIRS Hair abnormallyshort growth eg to enhance nutrient uptake ROOT Number Less root hairsthere is an Useful for increasing root HAIRS abnormally low growth eg toenhance number of root hairs nutrient uptake ROOT Other Other thiscorrelates with Useful for increasing root HAIRS any root hair mutantgrowth eg to enhance phenotypes which do nutrient uptake not fit intothe above categories (a picture should be taken for documentation) ROOTBulbous Root Bulbous Root Bulbous Root Hairs Useful for increasing rootHAIRS Hairs Hairs growth eg to enhance nutrient uptake ROOT BeardedBearded the lateral roots are Useful for increasing root (Nitrogen)(Nitrogen) long in high nitrogen, growth eg to enhance and they areshort in nutrient uptake low nitrogen PRIMARY Thickness Thicker Primarythe primary root is Useful for increasing root ROOT Root abnormallythick growth eg to enhance nutrient uptake WHOLE Stress Root Identifyplants with Useful for increasing root PLANT Architecture increased rootmass growth eg to enhance nutrient uptake PRIMARY Thickness ThinnerPrimary the primary root is Useful for increasing root ROOT Rootabnormally thin growth eg to enhance nutrient uptake PRIMARY Wavy Wavythere is a consistent Useful for increasing root ROOT and gentle wavygrowth eg to enhance appearance nutrient uptake LATERAL Length LongerLateral the lateral roots are Useful for increasing root ROOTS Rootabnormally long growth eg to enhance nutrient uptake LATERAL Number MoreLateral there is an Useful for increasing root ROOTS Roots abnormallyhigh growth eg to enhance number of lateral nutrient uptake roots ROOTNumber More root hairs there is an Useful for increasing root HAIRSabnormally high growth eg to enhance number of root hairs nutrientuptake Useful for increasing seed carbon or nitrogen SEED Seed WeightWeight weight of seed Useful for increasing seed weight SILIQUES LengthLong siliques are Useful for increasing abnormally long (the seed/fruityield or percent difference in modifying fruit content length comparedto the control should be noted in the comments) SILIQUES Length Shortsiliques are Useful for increasing abnormally short seed/fruit yield or(the percent modifying fruit content difference in length compared tothe control should be noted in the comments) SILIQUES Other Other thiscorrelates with Useful for increasing any silique mutant seed/fruityield or phenotypes which do modifying fruit content not fit into theabove categories (a picture should be taken for documentation) ROSETTESize Large rosette leaves are Useful for increasing LEAVES abnormallylarge vegetative growth and (the percent enhancing foliage difference insize compared to the control should be noted in the comments) Useful formaking nutraceuticals/pharmaceuticals in plants HYPOCOTYL Other Otherthis correlates with Useful for making larger any hypocotyl mutantplants phenotypes which do not fit into the above categories (a pictureshould be taken for documentation) WHOLE Other Other this correlateswith Useful for making larger SEEDLING any whole plant plants mutantphenotypes which do not fit into the above categories (a picture shouldbe taken for documentation) WHOLE Other Other this correlates withUseful for making larger PLANT any whole plant plants mutant phenotypeswhich do not fit into the above categories (a picture should be takenfor documentation) CAULINE Petiole Length Long Petioles the caulinepetioles Useful for making larger LEAVES are abnormally long plants (thepercent difference in size compared to the control should be noted inthe comments) WHOLE Size Large plant is abnormally Useful for makinglarger SEEDLING large (the percent plants difference in size compared tothe control should be noted in the comments) WHOLE Size Large plant isabnormally Useful for making larger PLANT large (the percent plantsdifference in size compared to the control should be noted in thecomments) SEED Lethal Lethal the seed is inviable Useful for makinglethal and appears as a plants for genetic small, dark, raisin-confinement systems like seed in the mature siligue WHOLE Germination NoGermination none of the seed Useful for making lethal SEEDLINGgerminates plants for genetic confinement systems WHOLE Germination Poora portion of the seed Useful for making lethal SEEDLING Germinationnever germinates plants for genetic confinement systems WHOLEGermination Slow a portion of the seed Useful for making lethal SEEDLINGGermination germinates plants for genetic significantly laterconfinement systems than the rest of the seed in the pot ROSETTEVitrified Vitrified leaves are somewhat Useful for making lethal LEAVEStranslucent or ?water plants for genetic soaked? confinement systemsCAULINE Vitrified Vitrified leaves are somewhat Useful for making lethalLEAVES translucent or ?water plants for genetic soaked? confinementsystems COTYLEDONS Albino Opaque Albino plant is opaque and Useful formaking lethal devoid of pigment plants for genetic confinement systemsCOTYLEDONS Albino Translucent plant is translucent Useful for makinglethal Albino and devoid of plants for genetic pigment confinementsystems WHOLE Lethal Seedling Lethal cotyledons emerge Useful for makinglethal SEEDLING (although they are plants for genetic often small), butthen confinement systems the plant ceases to develop further; No trueleaves appear and the plant dies early (These differ from yellow-greenlethals in that the cotyledons are wild- type in color and may not lookdiffer WHOLE Lethal Yellow-Green cotyledons are small Useful for makinglethal SEEDLING Lethal and pale yellow- plants for genetic green incolor, but confinement systems NOT totally devoid of pigment; Inaddition to yellow- green cotyledons, these plants produce no orseverely reduced size true leaves, which, if present, are alsoyellow-green; These plants die prem WHOLE Meristem Mutant MeristemMutant this term Useful for making lethal SEEDLING encompasses a plantsfor genetic variety of confinement systems phenotypes, all of which haveone thing in common, i.e., they all have something significantly wrongwith how the meristem is producing its leaves; Depending on the severityof the phenotype, the plants in this category WHOLE Seedling Seedlingthis term Useful for making lethal SEEDLING Defective Defectiveencompasses a plants for genetic variety of phenotypes confinementsystems which share similar characteristics, i.e., they are small, havedistorted structures, and are prone to early death; For example,patterning mutants would be a class of mutants which fall under thiscategory WHOLE Color Yellow-Green the leaves and Useful for makinglethal PLANT Viable 1 cotyledons are plants for genetic yellow-green inconfinement systems color, but this is not a lethal phenotype WHOLEColor Yellow-Green the leaves are yellow- Useful for making lethal PLANTViable 2 green in color but the plants for genetic cotyledons are aconfinement systems wild-type green in color WHOLE Color Yellow-Greenthe leaves start out Useful for making lethal PLANT Viable 3 wild-typegreen and plants for genetic gradually turn confinement systemsyellow-green in color, while the cotyledons stay wild- type green WHOLEColor Yellow-Green the leaves appear Useful for making lethal PLANTViable 4 wild-type green, but plants for genetic slowly turn yellow-confinement systems green over time, while the cotyledons appear andremain yellow-green WHOLE Stress Seed Bleaching Identify plants whoseUseful for making low PLANT seed coats do not fiber seeds with increasedbleach out under long digestability bleach soaking ROSETTE Fused LeafFused to the leaf is fused to an Useful for making LEAVES Inflorescenceinflorescence ornamental plants with flowers and leaves fused ROSETTEInterveinal Interveinal the leaf tissue is Useful for making LEAVESChlorosis Chlorosis chlorotic between its ornamental plants with veinsmodified color CAULINE Interveinal Interveinal the leaf tissue is Usefulfor making LEAVES Chlorosis Chlorosis chlorotic between its ornamentalplants with veins modified color FLOWER Organ Fused Sepals the sepalsare fused Useful for making Morphology together and won?t ornamentalplants with open naturally, but modified flowers the flower is otherwisewild-type FLOWER Organ Narrow Petals the petals are Useful for makingMorphology abnormally narrow ornamental plants with modified flowersFLOWER Organ Narrow Sepals the sepals are Useful for making Morphologyabnormally narrow ornamental plants with modified flowers FLOWER OrganShort Petals the petals are Useful for making Morphology abnormallyshort ornamental plants with modified flowers FLOWER Organ Short Sepalsthe sepals are Useful for making Morphology abnormally short ornamentalplants with modified flowers FLOWER Size Large flower is abnormallyUseful for making large (the percent ornamental plants with differencein size modified flowers compared to the control should be noted in thecomments) FLOWER Size Small flower is abnormally Useful for making small(the percent ornamental plants with difference in size modified flowerscompared to the control should be noted in the comments) FLOWER OtherOther this correlates with Useful for making any flower mutantornamental plants with phenotypes which do modified flowers not fit intothe above categories (a picture should be taken for documentation)INFLORESCENCE Aerial Rosette Aerial Fosette rosette forms at or Usefulfor making above the first ornamental plants with internode modifiedflowers INFLORESCENCE Appearance Corkscrew the inflorescence is Usefulfor making Appearance really twisted, almost ornamental plants with likea corkscrew, but modified flowers somewhat more irregular INFLORESCENCEAppearance Curved the inflorescence has Useful for making Appearance aslight, irregular ornamental plants with curve upwards, modified flowersgreater than that of the control plants INFLORESCENCE Appearance Multi-the inflorescence is Useful for making Inflorescence fused to anotherornamental plants with Fusion inflorescence, modified flowers creating acelery-like appearance INFLORESCENCE Appearance Undulate theinflorescence is Useful for making Appearance wavy in appearanceornamental plants with modified flowers INFLORESCENCE Branching Acaulinefirst branching is not Useful for making Branching subtended by aornamental plants with cauline leaf modified flowers INFLORESCENCE WaxGlaucous inflorescence is Useful for making abnormally dull inornamental plants with appearance modified flowers INFLORESCENCE WaxGlossy inflorescence is Useful for making shiny/glossy in ornamentalplants with appearance modified flowers INFLORESCENCE Other Other thiscorrelates with Useful for making any inflorescence ornamental plantswith mutant phenotypes modified flowers which do not fit into the abovecategories (a picture should be taken for documentation) COTYLEDONSAsymmetric Asymmetric the shape of the Useful for making cotyledon isornamental plants with asymmetric in modified foliage reference to thevertical axis ROSETTE Other Other this correlates with Useful for makingLEAVES any leaf mutant ornamental plants with phenotypes which domodified leaves not fit into the above categories (a picture should betaken for documentation) CAULINE Other Other this correlates with Usefulfor making LEAVES any cauline mutant ornamental plants with phenotypeswhich do modified leaves not fit into the above categories (a pictureshould be taken for documentation) FLOWER Homeotic Homeotic the flowerhas one or Useful for making plants Mutant Mutant more of its organssterile and for genetic converted to another confinement type of organ(specific details should be noted in the comments) FLOWER Organ AberrantOrgan there is an abnormal Useful for making plants Morphology Numbernumber of some or sterile and for genetic all of the flowers confinementorgans FLOWER Organ Short Stamens the stamens are Useful for makingplants Morphology abnormally short; sterile and for genetic This oftenleads to confinement mechanical problems with fertility FLOWER FertilityAborted fertility the ovule is Useful for making plants unfertilized andsterile and for genetic appears as a brown or confinement white speck inthe mature silique FLOWER Fertility Female-sterile there is a problemUseful for making plants with the ovules such sterile and for geneticthat no fertilization is confinement occurring FLOWER FertilityMale-sterile there is a problem Useful for making plants with the pollensuch sterile and for genetic that no fertilization is confinementoccurring FLOWER Fertility Reduced fertility a reduced number of Usefulfor making plants successful sterile and for genetic fertilizationevents, confinement and therefore seeds, are being produced by the plantFLOWER Fertility Sterile no successful Useful for making plantsfertilization events, sterile and for genetic and therefore no seedconfinement is being produced by the plant; The reason for thissterility is not known at the time of the observation FLOWER FertilityOther this correlates with Useful for making plants any fertility mutantsterile and for genetic phenotypes which do confinement not fit into theabove categories (a picture should be taken for documentation) WHOLEStress Early Flowering Identify plants that Useful for making plantsPLANT flower early that flower early COTYLEDONS Petiole Length LongPetioles the cotyledon petioles Useful for making plants are abnormallylong that grow and better in (the percent shade difference in sizecompared to the control should be noted in the comments) ROSETTE PetioleLength Varying Petiole the leaf petioles vary Useful for making plantsLEAVES Lengths in length throughout that grow better in shade therosette ROSETTE Petiole Length Long Petioles the leaf petioles areUseful for making plants LEAVES abnormally long (the that grow better inshade percent difference in size compared to the control should be notedin the comments) Useful for making plants tolerant to biotic stressWHOLE Stress Identify plants able to Useful for making plants PLANTtolerate high density tolerant to density and and no phosphate and lowfertilizer nitrogen, possible lead assay for vigor under populationdensity and low nutrient conditions WHOLE Stress pH (high) Identifyplants Useful for making plants PLANT tolerant to high pH, tolerant tohigh pH or low and possibly low phosphate phosphate WHOLE Stress LowNitrate Identify plants Useful for making plants PLANT tolerant to lowtolerant to low nitrogen nitrogen/nitrate growth media WHOLE StressLNABA Identify plants Useful for making plants PLANT tolerant to lowtolerant to low nitrogen nitrogen and high ABA concentrations WHOLEStress No Nitrogen Identify plants with Useful for making plants PLANTincreased vigor under tolerant to low nitrogen no nitrogen conditionsWHOLE Stress MSX Identify plants Useful for making plants PLANT tolerantto nitrogen tolerant to low nitrogen assimilation inhibitor, andpossibly low nitrogen tolerance and/or seed nitrogen accumulation WHOLEStress No N, No PO4 Identify plants Useful for making plants PLANTtolerant to no tolerant to low nitrogen and no nitrogen/low phosphatephosphate growth media WHOLE Stress Oxidative Identify plants Useful formaking plants PLANT tolerant to oxidative tolerant to oxidative stressstresses ROSETTE Trichomes Few Trichomes trichomes are sparse Useful formaking plants LEAVES but present on the with enhanced chemical leavescomposition ROSETTE Trichomes Glabrous trichomes are totally Useful formaking plants LEAVES absent with enhanced chemical composition ROSETTETrichomes Abnormal the trichomes are Useful for making plants LEAVESTrichome Shape abnormally shaped with enhanced chemical compositionCAULINE Trichomes Few Trichomes trichomes are sparse Useful for makingplants LEAVES but present on the with enhanced chemical leavescomposition CAULINE Trichomes Glabrous trichomes are totally Useful formaking plants LEAVES absent with enhanced chemical composition CAULINETrichomes Abnormal the trichomes are Useful for making plants LEAVESTrichome Shape abnormally shaped with enhanced chemical compositionINFLORESCENCE Trichomes Glabrous trichomes are totally Useful for makingplants absent with enhanced chemical composition INFLORESCENCE TrichomesAbnormal the trichomes are Useful for making plants Trichome Shapeabnormally shaped with enhanced chemical composition ROSETTE CurledCorkscrew leaves appear as Useful for making plants LEAVES “Curled 5”,with the with altered leaf shape eg additional attribute of curledleaves twisting like a corkscrew, instead of uniformly curling from bothsides of the leaf ROSETTE Curled Cup-shaped leaves are curled up Usefulfor making plants LEAVES at the leaf margins with altered leaf shape egsuch that they form a curled leaves cup or bowl-like shape ROSETTECurled Curled 1 leaves are abnormally Useful for making plants LEAVEScurled slightly up or with altered leaf shape eg down at the leaf curledleaves margins, but do not fall under the “cup- shaped” description(least severe type) ROSETTE Curled Curled 2 leaves are abnormally Usefulfor making plants LEAVES curled up or down at with altered leaf shape egthe leaf margins, but curled leaves do not fall under the “cup-shaped”description (more severe than Curled 1, but less severe than Curled 3)ROSETTE Curled Curled 3 leaves are abnormally Useful for making plantsLEAVES curled up or down at with altered leaf shape eg the leaf margins,but curled leaves do not fall under the “cup-shaped” description (moresevere than Curled 2, but less severe than Curled 4) ROSETTE CurledCurled 4 leaves are abnormally Useful for making plants LEAVEScurled/rolled up or with altered leaf shape eg down at the leaf curledleaves margins (more severe than Curled 3, but less severe than Curled5) ROSETTE Curled Curled 5 leaves are completely Useful for makingplants LEAVES curled/rolled up or with altered leaf shape eg down at theleaf curled leaves margins (most severe type) CAULINE Curled Corkscrewleaves appear as Useful for making plants LEAVES “Curled 5”, with thewith altered leaf shape eg additional attribute of curled leavestwisting like a corkscrew, instead of uniformly curling from both sidesof the leaf CAULINE Curled Cup-shaped the cauline leaves are Useful formaking plants LEAVES curled up at the leaf with altered leaf shape egmargins such that curled leaves they form a cup or bowl-like shapeCAULINE Curled Curled 1 the cauline leaves are Useful for making plantsLEAVES abnormally curled with altered leaf shape eg slightly up or downat curled leaves the leaf margins, but do not fall under the“cup-shaped” description (least severe type) CAULINE Curled Curled 2 thecauline leaves are Useful for making plants LEAVES abnormally curled upwith altered leaf shape eg or down at the leaf curled leaves margins,but do not fall under the “cup- shaped” description (more severe thanCurled 1, but less severe than Curled 3) CAULINE Curled Curled 3 thecauline leaves are Useful for making plants LEAVES abnormally curled upwith altered leaf shape eg or down at the leaf curled leaves margins,but do not fall under the “cup- shaped” description (more severe thanCurled 2, but less severe than Curled 4) CAULINE Curled Curled 4 thecauline leaves are Useful for making plants LEAVES abnormally withaltered leaf shape eg curled/rolled up or curled leaves down at the leafmargins (more severe than Curled 3, but less severe than Curled 5)CAULINE Curled Curled 5 the cauline leaves are Useful for making plantsLEAVES completely with altered leaf shape eg curled/rolled up or curledleaves down at the leaf margins (most severe type) ROSETTE Size Smallrosette leaves are Useful for making plants LEAVES abnormally small withdecreased vegetative (the percent growth difference in size compared tothe control should be noted in the comments) COTYLEDONS Wilted Wiltedcotyledons appear Useful for making plants wilted, i.e., they look withenhanced abiotic as though they have stress tolerance suffered fromdrought conditions ROSETTE Wax Glaucous leaves are abnormally Useful formaking plants LEAVES dull in appearance with enhanced abiotic stresstolerance ROSETTE Wax Glossy leaves are Useful for making plants LEAVESshiny/glossy in with enhanced abiotic appearance stress toleranceCAULINE Wax Glaucous leaves are abnormally Useful for making plantsLEAVES dull in appearance with enhanced abiotic stress tolerance CAULINEWax Glossy leaves are Useful for making plants LEAVES shiny/glossy inwith enhanced abiotic appearance stress tolerance WHOLE Stress MetabolicIdentify plants with Useful for making plants PLANT Profiling alteredmetabolic with enhanced metabolite profiles as defined in accumulation4a WHOLE Stress Plant Identify plants with Useful for making plantsPLANT Architecture improved architecture with enhanced plantarchitecture WHOLE Stress ABA Identify plants Useful for making plantsPLANT tolerant to ABA, and with enhanced tolerance possibly drought todrought and/or other stresses WHOLE Stress Mannitol Identify plantsUseful for making plants PLANT tolerant to mannitol, with enhancedtolerance and possibly drought to drought stress WHOLE StressDessication Identify plants Useful for making plants PLANT tolerant towater loss, with enhanced tolerance possibly drought to drought stresstolerant WHOLE Stress High Sucrose Identify plants Useful for makingplants PLANT tolerant to high with enhanced tolerance sucrose conditionsto drought (possible Lead assay for C/N partitioning) WHOLE Stress HeatIdentify plants with Useful for making plants PLANT thermotolerance withenhanced tolerance to heat WHOLE Stress High Nitrogen Identify plantsUseful for making plants PLANT tolerant to high with enhanced tolerancenitrogen conditions to high nitrogen WHOLE Stress Etiolation Identifyplants with Useful for making plants PLANT increased vigor in the withenhanced tolerance dark to light stress ROSETTE DisorganizedDisorganized rosette leaves do not Useful for making plants LEAVESRosette Rosette appear in the normal with increased biomass fashion,i.e., their phyllotaxy may be abnormal or too many leaves may beemerging in comparison to the control INFLORESCENCE Phyllotaxy EvenPhyllotaxy a phyllotaxy mutant Useful for making plants whose newbranches with increased biomass emerge at exactly the same height aseach other, i.e., there is no internode between them COTYLEDONS ShapeElliptic Shape cotyledons are quite Useful for making plants narrow andpointed, with increased biomass more so than and foliage lanceolateROSETTE Fused Leaf Fused to the leaf is fused to its Useful for makingplants LEAVES Petiole petiole with increased biomass and foliage ROSETTEShape Cordate Shaped similar to ovate, Useful for making plants LEAVESexcept the leaf is not with increased biomass rounded at its base andfoliage ROSETTE Shape Elliptic Shaped leaves are quite Useful for makingplants LEAVES narrow and pointed, with increased biomass more so thatand foliage lanceolate ROSETTE Shape Lanceolate leaves are narrow andUseful for making plants LEAVES Shaped come to a dull point withincreased biomass at the apex and foliage ROSETTE Shape Lobed Shapedleaves have very deep Useful for making plants LEAVES and rounded withincreased biomass serrations, giving an and foliage appearance of manylobes forming the margins of the leaves ROSETTE Shape Oval Shaped leavesare much Useful for making plants LEAVES rounder than wild- withincreased biomass type and foliage ROSETTE Shape Ovate Shaped leaves arewider at Useful for making plants LEAVES base than at apex, withincreased biomass otherwise similar to and foliage wild-type ROSETTEShape Serrate Margins leaf margins have Useful for making plants LEAVESlittle ?teeth? on them, with increased biomass i.e., they are serratedand foliage ROSETTE Shape Trident Shaped leaves look Useful for makingplants LEAVES somewhat like a with increased biomass trident, i.e., theyhave and foliage a sharp point at the apex, and a sharp point on eachside ROSETTE Shape Undulate Shaped leaves are wavy Useful for makingplants LEAVES with increased biomass and foliage WHOLE Rosette ShapeBushy Rosette the different petioles Useful for making plants PLANTShaped have very varied with increased biomass liminal angles, givingand foliage the plant a very bushy appearance; This is often accompaniedby a “Disorganized Rosette” phenotype WHOLE Rosette Shape Flat Rosettethe petioles have a Useful for making plants PLANT Shaped very smallliminal with increased biomass angle, i.e., the rosette and foliageappears flat instead of having its usual slight vertical angle WHOLERosette Shape Standing Rosette the petioles have a Useful for makingplants PLANT Shaped very large liminal with increased biomass angle,i.e., it appears and foliage as though the leaves are standing upinstead of having their usual small vertical angle from the soil CAULINEFused Leaf Fused to the cauline leaf is Useful for making plants LEAVESInflorescence fused to an with increased biomass inflorescence or andfoliage branch CAULINE Fused Leaf Fused to the cauline leaf is Usefulfor making plants LEAVES Leaf fused to itself or with increased biomassanother cauline leaf and foliage CAULINE Shape Cordate Shaped similar toovate, Useful for making plants LEAVES except the leaf is not withincreased biomass rounded at its base and foliage CAULINE Shape EllipticShaped leaves are quite Useful for making plants LEAVES narrow andpointed, with increased biomass more so that and foliage lanceolateCAULINE Shape Lanceolate leaves are narrow and Useful for making plantsLEAVES Shaped come to a dull point with increased biomass at the apexand foliage CAULINE Shape Lobed Shaped leaves have very deep Useful formaking plants LEAVES and rounded with increased biomass serrations,giving an and foliage appearance of many lobes forming the margins ofthe leaves CAULINE Shape Oval Shaped leaves are much Useful for makingplants LEAVES rounder than wild- with increased biomass type and foliageCAULINE Shape Ovate Shaped leaves are wider at Useful for making plantsLEAVES base than at apex, with increased biomass otherwise similar toand foliage wild-type CAULINE Shape Serrate Margins leaf margins haveUseful for making plants LEAVES little ?teeth? on them, with increasedbiomass i.e., they are serrated and foliage CAULINE Shape Trident Shapedleaves look Useful for making plants LEAVES somewhat like a withincreased biomass trident, i.e., they have and foliage a sharp point atthe apex, and a sharp point on each side CAULINE Shape Undulate Shapedleaves are wavy Useful for making plants LEAVES with increased biomassand foliage CAULINE Size Large cauline is abnormally Useful for makingplants LEAVES large (the percent with increased biomass difference insize and foliage compared to the control should be noted in thecomments) CAULINE Size Small cauline is abnormally Useful for makingplants LEAVES small (the percent with increased biomass difference insize and foliage compared to the control should be noted in thecomments) LATERAL Length Smaller Lateral the lateral roots are Usefulfor making plants ROOTS Root abnormally short with increased root growthto prevent lodging or enhance nutrient uptake PRIMARY Length LongPrimary the primary root is Useful for making plants ROOT Rootabnormally long with increased root (the percent growth to preventlodging difference in size or enhance nutrient compared to the uptakecontrol should be noted in the comments) PRIMARY Length Short Primarythe primary root is Useful for making plants ROOT Root abnormally shortwith increased root (the percent growth to prevent lodging difference insize or enhance nutrient compared to the uptake control should be notedin the comments) WHOLE Stress Plant Size Identify plants of Useful formaking plants PLANT increased size with increased size and compared towild biomass type WHOLE Stress Starch Identify plants with Useful formaking plants PLANT increased starch with increased starch accumulationcontent WHOLE Stress Cold Identify plants that Useful for making plantsPLANT Germination germinate better at with increased tolerance coldtemperatures to cold stress WHOLE Stress Cold Growth Identify plantsthat Useful for making plants PLANT grow faster at cold with increasedtolerance temperatures to cold stress WHOLE Stress Soil Drought Identifyplants with Useful for making plants PLANT increased tolerance to withincreased tolerance soil drought to drought WHOLE Stress Soil Drought -Identify plants that Useful for making plants PLANT Desiccation aretolerant to low with increased tolerance tolerance soil moisture and todrought resist wilting WHOLE Stress PEG Identify plants Useful formaking plants PLANT tolerant to PEG, and with increased tolerancepossibly drought to drought stress SEED Size Large the seed is Usefulfor making plants abnormally large with larger seeds (the percentdifference in size compared to the control should be noted in thecomments) INFLORESCENCE Branching Asecondary the plant does not Usefulfor making plants Branching form any secondary with modified flowersinflorescences SEED Size Small the seed is Useful for making plantsabnormally small with smaller seeds or no (the percent seeds differencein size compared to the control should be noted in the comments) WHOLEStress C/N Content Identify plants/seeds Useful for making seeds PLANTwith altered with altered carbon/nitrogen carbon/nitrogen levels levelsINFLORESCENCE Internode Length Short Internode the internode is Usefulfor making shorter abnormally short plants and plants with (the percentmodified flowers difference in length compared to the control should benoted in the comments) WHOLE Dwarf Brassino-Steroid these plants aresmall Useful for making smaller PLANT Dwarf in stature, dark green,plants have oval leaves, strong bolts, and are often sterile WHOLE DwarfMisc. Dwarf these are dwarf plants Useful for making smaller PLANT thedo not fall under plants the brassino-steroid dwarf category HYPOCOTYLLength Short hypocotyl is visibly Useful for making smaller shorter thanin wild- plants type (the percent difference in size compared to thecontrol should be noted in the comments) INFLORESCENCE Height Short theinflorescences of Useful for making smaller the plants are plantsabnormally short (plant height is encompassed under the whole plant sizecategory, but this entry would be used if the height of the plant isabnormal, but is otherwise of normal size) (the percent difference insize WHOLE Size Small plant is abnormally Useful for making smallerSEEDLING small (the percent plants difference in size compared to thecontrol should be noted in the comments) ROSETTE Petiole Length ShortPetioles the leaf petioles are Useful for making smaller LEAVESabnormally short plants (the percent difference in size compared to thecontrol should be noted in the comments) WHOLE Size Small plant isabnormally Useful for making smaller PLANT small (the percent plantsdifference in size compared to the control should be noted in thecomments) CAULINE Petiole Length Short Petioles the cauline petiolesUseful for making smaller LEAVES are abnormally short plants (thepercent difference in size compared to the control should be noted inthe comments) INFLORESCENCE Strength Strong the primary Useful formaking inflorescence appears stronger plants significantly stronger,whether by thickness or rigidity INFLORESCENCE Strength Weak the primaryUseful for making inflorescence appears stronger plants significantlyweaker, whether by thickness or rigidity INFLORESCENCE InflorescenceThickness thickness of the Useful for making primary inflorescencestronger plants HYPOCOTYL Length Long hypocotyl is visibly Useful formaking taller longer than in wild- plants type (the percent differencein size compared to the control should be noted in the comments)INFLORESCENCE Internode Length Long Internode the internode is Usefulfor making taller abnormally long (the plants and plants with percentdifference in longer flowers length compared to the control should benoted in the comments) INFLORESCENCE Height Tall the inflorescences ofUseful for making taller the plants are plants and plants withabnormally long longer inflorescences (plant height is encompassed underthe whole plant size category, but this entry would be used if theheight of the plant is abnormal, but is otherwise of normal size) (thepercent difference in size SEED Color Dark Color the seed is Useful formodifying abnormally dark fiber content in seed SEED Color Light Colorthe seed is Useful for modifying abnormally light; fiber content in seedTransparent Testa is an example of this phenotype SILIQUES Shape Bentthe silique has sharp Useful for modifying fruit bend to it part of theshape, composition and way down the length seed yield of the silique;this bend can be as much as approaching 90 degrees SILIQUES ShapeBulging the seeds in the Useful for modifying fruit silique appearsshape, composition and “shrink-wrapped”, seed yield giving the silique abulging appearance SILIQUES Shape Clubbed the silique is Useful formodifying fruit somewhat bulbous at shape, composition and its terminalend seed yield SILIQUES Shape Sickle the silique is curved, Useful formodifying fruit much like the blade shape, composition and of a sickleseed yield INFLORESCENCE Branching No Branching there is no branchingUseful for modifying at all plant architecture, ie amount of branchingINFLORESCENCE Branching Horizontal new branches arise at Useful formodifying Branching a 90 degree angle plant architecture, ie from thebolt they are branch angle emerging from COTYLEDONS HorizontallyHorizontally cotyledon is visibly Useful for modifying Oblong Oblongwider than it is long, plant architecture, ie leaf and it is alsostructure symmetrical (or very close to it) when cut along itshorizontal axis INFLORESCENCE Branching Two Leaf two cauline leavesUseful for modifying Branching subtend branches plant architecture, ieinstead of one reducing foliage INFLORESCENCE Branching Reduced Apicalthe dominance of the Useful for modifying Dominance primaryinflorescence plant structure, ie is diminished, with increasedbranching the secondaries appearing as dominant or nearly as dominantSEED Seed Stacked the seeds/embryos Useful for modifying seedArrangement Arrangement are stacked one on content top of the otherwithin the silique, instead of having the usual side-by-sidedistribution SEED Other Other this correlates with Useful for modifyingseed any seed mutant content phenotypes which do not fit into the abovecategories (a picture should be taken for documentation) SEED Shape OvalShape the seeds are much Useful for modifying seed more rounded on thestructure and composition ends, giving the seed a true oval appearanceSEED Shape Ridged Shape the seeds have small Useful for modifying seedridges or bumps on structure and composition them SEED Shape TaperedShape the ends of the seeds Useful for modifying seed narrow down to astructure and composition much sharper point than usual COTYLEDONSCotyledon Single Cotyledon Only one cotyledon Useful for modifying seedNumber appears after structure and content germination; This is simplyone cotyledon that had formed instead of two, and is not related to thefused phenotype; With this exception, the plant is often otherwisewild-type in appearance COTYLEDONS Cotyledon Tricot three cotyledonsUseful for modifying seed Number emerge instead of structure and contenttwo; With this exception, the plant is often otherwise wild- type inappearance COTYLEDONS Curled Cup-shaped cotyledons are curled Useful formodifying seed up at the cotyledon structure and content margins suchthat they form a cup or bowl-like shape COTYLEDONS Curled Curled 1cotyledons are Useful for modifying seed abnormally curled structure andcontent slightly up or down at the cotyledon margins, but do not fallunder the “cup- shaped” description (least severe type) COTYLEDONSCurled Curled 2 cotyledons are Useful for modifying seed abnormallycurled up structure and content or down at the cotyledon margins, but donot fall under the “cup-shaped” description (more severe than Curled 1,but less severe than Curled 3) COTYLEDONS Curled Curled 3 cotyledons areUseful for modifying seed abnormally curled up structure and content ordown at the cotyledon margins, but do not fall under the “cup-shaped”description (more severe than Curled 2, but less severe than Curled 4)COTYLEDONS Curled Curled 4 cotyledons are Useful for modifying seedabnormally structure and content curled/rolled up or down at thecotyledon margins (more severe than Curled 3, but less severe thanCurled 5) COTYLEDONS Curled Curled 5 cotyledons are Useful for modifyingseed completely structure and content curled/rolled up or down at thecotyledon margins (most severe type) COTYLEDONS Dimorphic Dimorphic onecotyledon is Useful for modifying seed Cotyledons Cotyledonssignificantly larger structure and content than the other COTYLEDONSFused Fused 1 cotyledons are fused Useful for modifying seed to eachother, structure and content creating one cotyledon structure (leastsevere type) COTYLEDONS Fused Fused 2 cotyledons are fused Useful formodifying seed to each other, structure and content creating onecotyledon structure (more severe than Fused 1, but less severe thanFused 3) COTYLEDONS Fused Fused 3 cotyledons are fused Useful formodifying seed to each other, structure and content creating onecotyledon structure (more severe than Fused 2, but less severe thanFused 4) COTYLEDONS Fused Fused 4 cotyledons are fused Useful formodifying seed to each other, structure and content creating onecotyledon structure (more severe than Fused 3, but less severe thanFused 5) COTYLEDONS Fused Fused 5 cotyledons are fused Useful formodifying seed to each other, structure and content creating onecotyledon structure (most severe type) COTYLEDONS Other Other thiscorrelates with Useful for modifying seed any cotyledon mutant structureand content phenotypes which do not fit into the above categories (apicture should be taken for documentation) ROSETTE Fused Leaf Fused tothe leaf is fused to Useful for plants with LEAVES Leaf itself oranother leaf fused leaves eg ornamentals COTYLEDONS Petiole Length ShortPetioles the cotyledon petioles Useful for shade are abnormally shortavoidance and for making (the percent smaller plants difference in sizecompared to the control should be noted in the comments) PRIMARYAgravitropic Agravitropic the primary root does ROOT not appear to havea gravitropic response PRIMARY Kinked Kinked there is a sharp bend ROOTin the root ROSETTE Rosette Diameter Diameter diameter of rosette LEAVESWHOLE Plant Weight Plant Weight weight of whole plant PLANT WHOLE PlantHeight Height height of whole plant PLANT WHOLE Plant DTH Dth days toharvest of PLANT plant WHOLE Plant Harvest Harvest Index harvest indexof plant PLANT Index CAULINE Fused Leaf Fused to the cauline leaf isLEAVES Petiole fused to its petiole N/A N/A N/A N/A WHOLE HERBICIDEHERBICIDE herbicide segregation PLANT SEGREGATION SEGREGATION ratioWHOLE N/A No Mutant The plants were PLANT Phenotype screened at allObserved appropriate stages and showed no mutant phenotype, i.e., theylooked like normal, wild type Arabidopsis plants

From the results reported in Table 1 and the Sequence Listing, it can beseen that the nucleotides/polypeptides of the inventions are useful,depending upon the respective individual sequence, to make plants withmodified growth and phenotype characteristics, including:

-   -   a. modulated plant size, including increased and decreased        height or length;    -   b. modulated vegetative growth (increased or decreased);    -   c. modulated organ number;    -   d. increased biomass;    -   e. sterility;    -   f. seedling lethality;    -   g. accelerated crop development or harvest;    -   h. accelerated flowering time;    -   i. delayed flowering time;    -   j. delayed senescence;    -   k. enhanced drought or stress tolerance;    -   l. increased chlorophyll and photosynthetic capacity;    -   m. increased anthocyanin content;    -   n. increased root growth, and increased nutrient uptake;    -   o. increased or decreased seed weight or size, increased seed        carbon or nitrogen content;    -   p. modified, including increased, seed/fruit yield or modified        fruit content;    -   q. enhanced foliage; r. usefulness for making        nutratceuticals/pharmaceuticals in plants;    -   s. plant lethality;    -   t. decrease seed fiber content to provide increased        digestability;    -   u. modified ornamental appearance with modified leaves, flowers,        color or foliage;    -   v. modified sterility in plants;    -   w. enhanced ability to grow in shade;    -   x. enhanced biotic stress tolerance;    -   y. increased tolerance to density and low fertilizer;    -   z. enhanced tolerance to high or low pH, to low or high nitrogen        or phosphate;    -   aa. enhanced tolerance to oxidative stress;    -   bb. enhanced chemical composition;    -   cc. altered leaf shape;    -   dd. enhanced abiotic stress tolerance;    -   ee. increased tolerance to cold stress;    -   ff. increased starch content;    -   gg. reduced number or no seeds;    -   hh. enhanced plant strength;    -   ii. modified flower length;    -   jj. longer inflorescences;    -   kk. modified seed fiber content;    -   ll. modified fruit shape;    -   mm. modified fruit composition;    -   nn. modified seed yield;    -   oo. modified plant architecture, such as modified amount or        angle of branching, modified leaf structure, or modified seed        structure; and    -   pp. enhanced shade avoidance.

EXAMPLE 1 Lead 80 (ME08386); Clone 733804 SEQ ID NO. 94

Lead 80 (SEQ ID NO. 94) encodes a 92 amino acid bHLH transcriptionfactor from wheat. Plants transformed with this sequence were found toexhibit:

-   -   Enhanced growth, particularly on low-nitrate-containing media;    -   Enhanced photosynthesis on low-nitrate containing media;    -   Elongated hyocotyls, narrow leaves and often a flattened        inflorescence.

Clone 733804 encodes a bHLH transcription factor that confers increasedgrowth and improved photosynthetic efficiency on plants experiencingnitrogen deficiency stress. Transcription factors often control theexpression of multiple genes in a pathway. As such, Clone 733804 may beinvolved in controlling the expression of several genes in a pathway,such as carbon flux through the TCA cycle (Yanagisawa et al., 2004). Arelated Arabidopsis bHLH transcription factor and potential ortholog(60% identity; clone 8607) is also able to confer a similar low nitrogenstress phenotype. Since the gain-of-function phenotype of clones 733804and clone 8607 is conserved between wheat and Arabidopsis, these genescan have direct application for improving nitrogen stress toleranceincreasing nitrogen use efficiency, and enhancing seedling vigor in abroad range of crops.

Material and Methods:

Generation and Phenotypic Evaluation of T₁ Events.

Wild-type Arabidopsis Wassilewskija (Ws) plants were transformed with aTi plasmid containing Clone 733804 in the sense orientation relative tothe 35S promoter, as described above. The Ti plasmid vector used forthis construct, CRS 338, contains the Ceres-constructed, plantselectable marker gene phosphinothricin acetyltransferase (PAT) whichconfers herbicide resistance to transformed plants. Ten independentlytransformed events were selected and evaluated for their qualitativephenotype in the T₁ generation.

The procedure for 1) identifying the candidate from a low nitratetolerance superpool screen, 2) confirming the phenotype in the secondand third generations and 3) determining the lack of significantnegative phenotypes was as described below.

Screening Superpools 62-71 for Tolerance to Low Nitrate GrowthConditions.

Two thousand seeds each from Superpools 62-71 were pooled together andplated on low nitrate media DNA was isolated from each candidate plantand sequenced to determine which transgene was present.

Growth Conditions and Planting Schema for ME08386 Under Low NitrateGrowth Conditions.

Evaluation of tolerance to low nitrate conditions (300 μM KNO₃ MS media)was done using five T₂ events (-01, -03, -04, -08 and -09).Subsequently, T₃ generation seeds for all five events were evaluatedunder low nitrate conditions.

Results:

ME08386 was Identified from a Superpool Screen for Seedling Tolerance toLow Nitrate Conditions.

Superpools 62-71 were screened for seedlings that were larger or greenerthan controls on low nitrate growth media. Transgene sequence wasobtained for 19 candidate seedlings. Two of the 19 candidate sequencesBLASTed to ME08386.

Four Events of ME08386 Show 3:1 Segregation for Finale™ Resistance.

Events -01, -04, -08 and -09 segregated 3:1 (R:S) for Finale™ resistancein the T₂ generation (data not shown).

Four Events of ME08386 Showed Significantly Increased Growth Under LowNitrate Growth Conditions in Both Generations.

Five events of ME08386 were sown as described in the Low Nitrate Assayin both the T₂ and the T₃ generations. In this study the seedling areaat 14 days for transgenic plants within an event was compared to theseedling area for non-transgenic segregants pooled across the sameplate. Four events, -01, -04, -08 and -09, were significant in bothgenerations at p=0.05, using a one-tailed t-test assuming unequalvariance (Table 1-1). TABLE 1-1 T-test comparison of seedling areabetween transgenic seedlings and pooled non-transgenic segregants after14 days of growth on low nitrate. Trans- genic Pooled Non- AvgTransgenics Line Events (cm²) n Avg (cm²) n t-test p-value ME08386ME08386-01 0.073 29 0.056 17 0.00132 ME08386 ME08386-01-99 0.086 320.056 17 9.83E−07 ME08386 ME08386-04 0.067 32 0.056 18 9.22E−05 ME08386ME08386-04-99 0.078 29 0.056 18 3.74E−06 ME08386 ME08386-08 0.064 270.053 20 0.0105  ME08386 ME08386-08-99 0.087 29 0.053 20 1.54E−09ME08386 ME08386-09 0.065 30 0.057 19 0.00549 ME08383 ME08386-09-99 0.07232 0.057 19 9.57E−07Four Events of ME08386 Showed Significantly Increased PhotosyntheticEfficiency under Low Nitrate Growth Conditions in Both Generations.

Five events of ME08386 were sown as described in the Low Nitrate Assayin both the T₂ and the T₃ generations. In this study, the seedlingphotosynthetic efficiency was measured as Fv/Fm comparing transgenicplants within an event to non-transgenic segregants pooled across thesame plate. Four events, -01, -04, -08 and -09, were significant in bothgenerations at p=0.05, using a one-tailed t-test assuming unequalvariance (Table 1-2). TABLE 1-2 T-test comparison of seedlingphotosynthetic efficiency between transgenic seedlings and poolednon-transgenic segregants after 14 days of growth on low nitrate. Trans-Pooled Non- genic Transgenics Line Events Fv/Fm n Fv/Fm n t-test p-valueME08386 ME08386-01 0.59 29 0.56 17 0.00982  ME08386 ME08386-01-99 0.6432 0.56 17 8.76E−07 ME08386 ME08386-04 0.61 31 0.56 19 0.00338  ME08386ME08386-04-99 0.61 28 0.56 19 0.00402  ME08386 ME08386-08 0.62 28 0.5221 4.69E−05 ME08386 ME08386-08-99 0.63 26 0.52 21 1.59E−09 ME08386ME08386-09 0.58 30 0.51 18 0.000819 ME08383 ME08386-09-99 0.58 32 0.5118 0.000357Qualitative Analysis of the T₁ Plants:

The physical appearance of four of the ten T₁ plants was identical tothe controls. Events -01, -03, -04, -08, -09 and -10 were noted ashaving flat inflorescences, but were still fully fertile.

Qualitative and Quantitative Analysis of the T₂ Plants (Screening forNegative Phenotypes):

Events -01, -04, -08 and -09 of ME08386 exhibited no statisticallyrelevant negative phenotypes. All four events showed the same flatinflorescence phenotype as noted in the T₁ generation, but thisphenotype does not negatively affect yield. The plants also had slightlyelongated hypocotyls and rosette leaves. The plants exhibited slightlyelongated hypocotyls, elongated rosette leaves and flat bolts. Butexhibited no observable or statistical differences between experimentalsand controls with respect to germination rate, days to flowering,rosette after 7 days post-bolting, or fertility (silique number and seedfill).

EXAMPLE 2 Lead 81 (ME03973) Clone 8607 SEQ ID NO. 96

Lead 81 (SEQ ID NO. **) encodes a 94 amino acid bHLH transcriptionfactor from Arabidopsis. Plants transformed with this sequence werefound to exhibit:

-   -   Enhanced growth, particularly on low-nitrate-containing media;    -   Enhanced photosynthesis on low-nitrate containing media;    -   Elongate hypocotyls, narrow leaves and often a flattened        inflorescence.

Clone 8607 encodes an Arabidopsis basic-helix-loop-helix transcriptionfactor. The clone was placed in the cDNA misexpression pipeline to testits utility in improving plant performance under various stressconditions. The gene is differentially expressed in heat, drought, andnitrogen-deficiency stress experiments and, therefore, can play a rolein regulating genes important for stress tolerance or adaptation.

Clone 8607 encodes a bHLH transcription factor that confers increasedgrowth and improved photosynthetic efficiency on plants experiencingnitrogen deficiency stress. Transcription factors often control theexpression of multiple genes in a pathway. Clone 8607 may be involved incontrolling the expression of several genes in a pathway, such as carbonflux through the TCA cycle (Yanagisawa et al., 2004). The function ofclone 8607 is not known, but its regulation by nitrogen stress indicatesit can function in plant responses to nitrogen deficiency. A relatedwheat bHLH transcription factor and potential ortholog (60% identity;clone 733804) is also able to confer a similar low nitrogen stressphenotype. Since the gain of function phenotype of clones 733804 andclone 8607 is conserved between wheat and Arabidopsis, these genes canhave direct application for improving nitrogen stress tolerance andincreasing nitrogen use efficiency and enhancing seedling vigor in abroad range of crops.

Materials and Methods:

Generation and Phenotypic Evaluation of T₁ Events.

Wild-type Arabidopsis Wassilewskija (Ws) plants were transformed with aTi plasmid containing Clone 8607 in the sense orientation relative tothe 35S promoter, as described above. The Ti plasmid vector used forthis construct, CRS 338, contains the Ceres-constructed, plantselectable marker gene phosphinothricin acetyltransferase (PAT) whichconfers herbicide resistance to transformed plants. Five independentlytransformed events were selected and evaluated for their qualitativephenotype in the T₁ generation as per Ceres SOP 5-HTP T1 PlantPhenotyping.

The procedure for 1) identifying the candidate from a low nitratetolerance superpool screen, 2) confirming the phenotype in the secondand third generations and 3) determining the lack of significantnegative phenotypes was as described below.

Screening Superpools 22-31 for Tolerance to Low Nitrate GrowthConditions.

Two thousand seeds each from Superpools 22-31 were pooled together andplated on low nitrate media DNA was isolated from each candidate plantand sequenced to determine which transgene was present.

Growth Conditions and Planting Schema for ME03973 Under Low NitrateGrowth Conditions.

Evaluation of tolerance to low nitrate conditions (300 μM KNO₃ MS media)was done using four T₂ events (-01, -02, -03 and -05). Subsequently, T₃generation seeds for all four events were evaluated under low nitrateconditions.

Results:

ME03973 was Identified from a Superpool Screen for Seedling Tolerance toLow Nitrate Conditions.

Superpool 27 was screened for seedlings that were larger or greener thancontrols on low nitrate growth media. Line ME03973 was identified fromamong the candidates.

Superpools 22-31 were screened for seedlings that were larger or greenerthan controls on low nitrate growth media. Transgene sequence wasobtained for 39 candidate seedlings. One of the 39 candidate sequencesBLASTed to ME03973.

Three Events of ME03973 Show 3:1 Segregation for Finale™ Resistance.

Events -01, -03 and -05 segregated 3:1 (R:S) for Finale™ resistance inthe T₂ generatio.

Three Events of ME03973 Showed Significantly Increased Growth Under LowNitrate Growth Conditions in Both Generations.

Four events of ME03973 were tested on the Low Nitrate Assay in both theT₂ and the T₃ generations. In this study, the seedling area at 14 daysfor transgenic plants within an event was compared to the seedling areafor non-transgenic segregants pooled across the same plate. Threeevents, -01, -03 and -05, were significant in both generations atp=0.05, using a one-tailed t-test assuming unequal variance (Table 2-1).TABLE 2-1 T-test comparison of seedling area between transgenicseedlings and pooled non-transgenic segregants after 14 days of growthon low nitrate. Trans- genic Pooled Non- Avg Transgenics Line Events(cm²) n Avg (cm²) n t-test p-value ME03973 ME03973-01 0.077 27 0.055 172.35E−08 ME03973 ME03973-01-99 0.083 35 0.055 17 3.20E−10 ME03973ME03973-03 0.073 33 0.056 11 0.000118 ME03973 ME03973-03-99 0.086 330.056 11 6.05E−06 ME03973 ME03973-05 0.069 29 0.061 20 0.0101 ME03973ME03973-05-99 0.077 29 0.061 20 1.69E−05Three Events of ME03973 Showed Significantly Increased PhotosyntheticEfficiency Under Low Nitrate Growth Conditions in Both Generations.

Four events of ME03973 were tested on the Low Nitrate Assay in both theT₂ and the T₃ generations. In this study, the seedling photosyntheticefficiency was measured as Fv/Fm comparing transgenic plants within anevent to non-transgenic segregants pooled across the same plate. Threeevents, -01, -03 and -05, were significant in both generations atp=0.05, using a one-tailed t-test assuming unequal variance (Table 2-2).TABLE 2-2 T-test comparison of seedling photosynthetic efficiencybetween transgenic seedlings and pooled non-transgenic segregants after14 days of growth on low nitrate. Trans- Pooled Non- genic TransgenicsLine Events Fv/Fm n Fv/Fm n t-test p-value ME03973 ME03973-01 0.61 270.56 17 0.000812 ME03973 ME03973-01-99 0.63 35 0.56 17 9.38E−06 ME03973ME03973-03 0.61 33 0.54 12 0.00131 ME03973 ME03973-03-99 0.63 33 0.54 124.11E−05 ME03973 ME03973-05 0.61 29 0.54 20 0.000375 ME03973ME03973-05-99 0.63 26 0.54 20 8.18E−06Qualitative Analysis of the T₁ Plants:

The noted physical appearance of the ten plants was identical to thecontrols. However, it is very likely that the elongated hypocotyls androsette leaves, and flattened inflorescence was phenotype was present inthe T₁ plants, but too subtle to be noted.

Qualitative and Quantitative Analysis of the T₂ Plants (Screening forNegative Phenotypes):

Events -01, -03 and -05 of ME03973 exhibited no statistically relevantnegative phenotypes. However, all events showed a flat inflorescencephenotype as noted in the T₁ generation, but this phenotype does notnegatively affect yield. The plants also had slightly elongatedhypocotyls and rosette leaves. The plants exhibited These events hadslightly elongated hypocotyls, elongated rosette leaves and flat bolts,but exhibited no observable or statistical differences betweenexperimentals and controls with respect to germination rate, days of theflowering, rosette area 7 days post-bolting, or fertility (siliquenumber and seed fill).

EXAMPLE 3 Lead 113 (ME08317): Clone 560948 SEQ ID NO. 90

Plant Construct Event/Generation Stage Assay Result 35S::560948 -01/T₂segregating Seedling Low Nitrate Significant plants Tolerance at p ≦ .0535S::560948 -05/T₂ segregating Seedling Low Nitrate Significant plantsTolerance at p ≦ .05 35S::560948 -01/T₃ segregating Seedling Low NitrateSignificant plants Tolerance at p ≦ .05 35S::560948 -05/T₃ segregatingSeedling Low Nitrate Significant plants Tolerance at p ≦ .05

Ectopic expression of Clone 560948 under the control of the 35S promoterresults in enhanced growth on low nitrate-containing media after 14 dayscompared to controls.

ME08317 is Homologous to Leads 80 & 81.

ME08317 was identified from a reciprocal BLAST algorithm as havingbetween 60-70% identity to Leads 80 & 81.

One Event of ME08317 Segregates for a Single Insert, While the OtherEvent Segregates for 2 Inserts.

Event -01 segregated 3:1 (R:S) for Finale™ resistance in the T₂generation. Event -05 segregated 15:1 (R:S) (data not shown).

Two Events of ME08317 Showed Significantly Enhanced Growth Under LowNitrate Growth Conditions in Both Generations.

Seeds representing two events of ME08317 from each of the T₂ and the T₃generations were sown under conditions described in the Low NitrateAssay. Both events, -01 and -05, showed a significant increase in growthin both generations at p=0.05 as measured using a one-tailed t-test andassuming unequal variance (Table 3-1). TABLE 3-1 T-test comparison ofseedling area between transgenic seedlings and pooled non-transgenicsegregants after 14 days of growth on low nitrate. Trans- genic PooledNon- Avg Transgenics Line Events (cm²) n Avg (cm²) n t-test p-valueME08317 ME08317-01 (T₂) 0.076 41 0.058 32 6.8 × 10⁻⁷ ME08317 ME08317-01(T₃) 0.074 27 0.058 32 1.7 × 10⁻⁸ ME08317 ME08317-05 (T₂) 0.081 47 0.05518 5.5 × 10⁻⁸ ME08317 ME08317-05 (T₃) 0.078 35 0.055 18 3.6 × 10⁻⁸Qualitative Analysis of the T₁ Plants:

All events appeared wild-type. It is possible the T₂ morphologicalphenotype below was present in the T₁ generation, but too subtle to benoted.

Qualitative and Quantitative Analysis of the T₂ Plants:

Events -01 and -05 of ME08317 had flat inflorescences and slightlyelongated hypocotyls and rosette leaves.

EXAMPLE 4 Lead 114 (ME10686); Clone 336524 (SEQ ID NO: 82)

Event/ Construct Generation Plant Stage Assay Result 35S::336524 -01/T₃Seedling Low Ammonium Significant segregating Nitrate Tolerance at p ≦.05 plants 35S::336524 -08/T₂ Seedling Low Ammonium Significantsegregating Nitrate Tolerance at p ≦ .05 plants 35S::336524 -01/T₄Seedling Low Ammonium Significant segregating Nitrate Tolerance at p ≦.05 plants 35S::336524 -08/T₃ Seedling Low Ammonium Significantsegregating Nitrate Tolerance at p ≦ .05 plants

Ectopic expression of Clone 336524 under the control of the 35S promoterresults in enhanced growth on low nitrate-containing media after 14 dayscompared to controls.

ME10686 is Homologous to Leads 80 & 81.

ME10686 was identified from a reciprocal BLAST algorithm as havingapproximately 60% identity to Leads 80 & 81.

Two Events of ME10686 Segregate for a Single Insert.

Events -01 and -08 segregated 3:1 (R:S) for Finale® resistance in the T₂generation (data not shown).

Two Events of ME10686 Showed Significantly Enhanced Growth Under LowAmmonium Nitrate Growth Conditions in Both Generations.

Seeds representing two events of ME10686 from each of the T₂ and the T₃generations (or T₃ and T₄ generations, as is the case for Event -01)were sown under conditions described in the Low Ammonium Nitrate Assay.Both events, -01 and -08, showed a significant increase in growth inboth generations at p=0.05 as measured using a one-tailed t-test andassuming unequal variance (Table 4-1). TABLE 4-1 T-test comparison ofseedling area between transgenic seedlings and pooled non-transgenicsegregants after 14 days of growth on low ammonium nitrate. Trans- genicPooled Non- Avg Transgenics Line Events (cm²) n Avg (cm²) n t-testp-value ME10686 ME10686-01 (T₃) 0.081 44 0.063 6 1.36 × 10⁻⁵ ME10686ME10686-01 (T₄) 0.078 50 0.063 6 9.06 × 10⁻⁵ ME10686 ME10686-08 (T₂)0.111 31 0.084 20 5.73 × 10⁻⁴ ME10686 ME10686-08 (T₃) 0.103 41 0.084 201.15 × 10⁻³

Qualitative Analysis of the T₁ Plants:

All events appeared wild-type. It is possible the T₂ morphologicalphenotype below was present in the T₁ generation, but too subtle to benoted.

Qualitative and Quantitative Analysis of the T₂ Plants:

Events -01 and -08 of ME08317 flat inflorescences and slightly elongatedhypocotyls and rosette leaves.

EXAMPLE 5 Lead ME08328: Clone 560681 (SEQ ID NO:88)

Event/ Construct Generation Plant Stage Assay Result 35S::560681 -05/T₂Seedling Low Ammonium Significant segregating Nitrate Tolerance at p ≦.05 plants 35S::560681 -05/T₂ Seedling Low Nitrate Significantsegregating Tolerance at p ≦ .05 plants

ME08328 was identified from a reciprocal BLAST algorithm as havingapproximately 70% identity to Leads 80 & 81.

ME08328-05 Segregates for a Single Insert.

Event -05 segregated 3:1 (R:S) for Finales resistance in the T₂generation (data not shown).

One Event of ME08328 Showed Significantly Enhanced Growth Under Both LowAmmonium Nitrate and Low Nitrate Growth Conditions.

Seeds representing one event of ME08328 were sown under conditionsdescribed in the Low Ammonium Nitrate and Low Nitrate Assays. Event -05showed a significant increase in growth in both generations at p=0.05 asmeasured using a one-tailed t-test and assuming unequal variance (Tables5-1 and 5-2). TABLE 5-1 T-test comparison of seedling area betweentransgenic seedlings and pooled non-transgenic segregants after 17 daysof growth on low ammonium nitrate. Trans- genic Pooled Non- AvgTransgenics Line Events (cm²) n Avg (cm²) n t-test p-value ME08328ME08328-05(T₂) 0.107 16 0.081 25 5.11 × 10⁻⁵

TABLE 5-2 T-test comparison of seedling area between transgenicseedlings and pooled non-transgenic segregants after 17 days of growthon low nitrate. Trans- genic Pooled Non- Avg Transgenics Line Events(cm²) n Avg (cm²) n t-test p-value ME08328 ME08328-05(T₂) 0.069 14 0.05718 0.048Qualitative Analysis of the T₁ Plants:

All events appeared wild-type.

EXAMPLE 6 Lead ME01905; Clone 4734 (SEQ ID NO: 84)

Event/ Construct Generation Plant Stage Assay Result 35S::4734 -03/T₂Seedling Low Ammonium Significant segregating Nitrate Tolerance at p ≦.05 plants 35S::4734 -05/T₂ Seedling Low Ammonium Significantsegregating Nitrate Tolerance at p ≦ .05 plants 35S::4734 -03/T₂Seedling Low Nitrate Significant segregating Tolerance at p ≦ .05 plants35S::4734 -05/T₂ Seedling Low Nitrate Significant segregating Toleranceat p ≦ .05 plantsME01905 is Homologous to Leads 80 & 81.

ME01905 was identified from a reciprocal BLAST algorithm as havingapproximately 60% identity to Leads 80 & 81.

Two Events of ME01905 Show 3:1 Segregation for Finale™ Resistance.

Events -03 and -05 segregated 3:1 (R:S) for Finale™ resistance in the T₂generation (data not shown).

Two Events of ME01905 Showed Significantly Enhanced Growth Under BothLow Ammonium Nitrate and Low Nitrate Growth Conditions.

Seeds representing two events of ME01905 were sown under conditionsdescribed in the Low Ammonium Nitrate and Low Nitrate Assays. Events -03and -05 showed a significant increase in growth in both generations atp=0.05 as measured using a one-tailed t-test and assuming unequalvariance (Tables 6-1 and 6-2). TABLE 6-1 T-test comparison of seedlingarea between transgenic seedlings and pooled non-transgenic segregantsafter 17 days of growth on low ammonium nitrate. Trans- genic PooledNon- Avg Transgenics Line Events (cm²) n Avg (cm²) n t-test p-valueME01905 ME08328-03(T₂) 0.119 13 0.088 34 0.0012 ME01905 ME08328-05(T₂)0.107 17 0.096 32 0.0041

TABLE 6-2 T-test comparison of seedling area between transgenicseedlings and pooled non-transgenic segregants after 17 days of growthon low nitrate. Trans- genic Pooled Non- Avg Transgenics Line Events(cm²) n Avg (cm²) n t-test p-value ME01905 ME08328-03(T₂) 0.086 12 0.05831 1.58 × 10⁻⁵ ME01905 ME08328-05(T₂) 0.075 17 0.06 33 8.91 × 10⁻⁵Qualitative Analysis of the T₁ Plants:

Events -01, —O₂, -03 and -05 had flat inflorescences, but were stillfully fertile. Event -03 was also noted as having a glossy appearance.

Qualitative and Quantitative Analysis of the T₂ Plants:

Events -01, -02, -03 and -05 of ME01905 had flat inflorescences andslightly elongated hypocotyls and rosette leaves. Events -01, -03 and-05 had a smaller rosette size and less seed yield compared to thecontrol. Event -02 had a normal rosette size and seed yield.

EXAMPLE 7 Lead ME01770: Clone 519 (SEQ ID NO: 86)

Event/ Construct Generation Plant Stage Assay Result 35S::519 -02/T₃Seedling Low Ammonium Significant segregating Nitrate Tolerance at p ≦.05 plants 35S::4734 -07/T₃ Seedling Low Ammonium Significantsegregating Nitrate Tolerance at p ≦ .05 plants 35S::519 -02/T₃ SeedlingLow Nitrate Significant segregating Tolerance at p ≦ .05 plants35S::4734 -07/T₃ Seedling Low Nitrate Significant segregating Toleranceat p ≦ .05 plantsME01770 is Homologous to Leads 80 & 81.

ME01770 was identified from a reciprocal BLAST algorithm as havingapproximately 70% identity to Leads 80 & 81.]

Two Events of ME01770 Show 3:1 Segregation for Finale™ Resistance.

Events -02 and -07 segregated 3:1 (R:S) for Finale™ resistance in the T₂generation (data not shown).

Two Events of ME01770 Showed Significantly Enhanced Growth Under BothLow Ammonium Nitrate and Low Nitrate Growth Conditions.

Seeds representing two events of ME01770 were sown under conditionsdescribed in the Low Ammonium Nitrate and Low Nitrate Assays. Events -02and -07 showed a significant increase in growth in both generations atp=0.05 as measured using a one-tailed t-test and assuming unequalvariance (Tables 7-1 and 7-2). TABLE 7-1 T-test comparison of seedlingarea between transgenic seedlings and pooled non-transgenic segregantsafter 17 days of growth on low ammonium nitrate. Pooled Non- TransgenicTransgenics t-test Line Events Avg (cm²) n Avg (cm²) n p-value ME01770ME01770- 0.125 12 0.106 17 0.0025 02(T₃) ME01770 ME01770- 0.121 11 0.08834 1.74 × 10⁻⁴ 07(T₃)

TABLE 7-2 T-test comparison of seedling area between transgenicseedlings and pooled non-transgenic segregants after 17 days of growthon low nitrate. Pooled Non- Transgenic Transgenics t-test Line EventsAvg (cm²) n Avg (cm²) n p-value ME01770 ME01770- 0.115 12 0.083 11 5.85× 10⁻⁴ 02(T₃) ME01770 ME01770- 0.083 10 0.058 31 0.0013 07(T₃)

Qualitative Analysis of the T₁ Plants:

Event -01 was small with a long hypocotyl and died before flowering.Events -08 and -09 had long hypocotyls and died before flowering. Events-03 and -04 were small. Events -02 and -05 had long hypocotyls. Events-06 and -07 were small with long hypocotyls.

Qualitative and Quantitative Analysis of the T₂ Plants:

Events -02, -04, -05, -06 and -07 of ME01770 had flat inflorescences andslightly elongated hypocotyls and rosette leaves. These events also hadsmaller rosettes and less seed yield compared to controls.

EXAMPLE 8 Lead ME21445: Clone 653656 (SEQ ID NO: 92)

Construct Generation Plant Stage Assay Result 326::653656 T₁ plantsSeedling Morphological Significant PhenotypingME21445 is Homologous to Leads 80 & 81.

ME21445 was identified from a reciprocal BLAST algorithm as havingapproximately 80% identity to Leads 80 & 81.

Multiple Events of ME21445 Showed Significantly Enhanced Growth as T₁Seedlings, with No Apparent Negative Phenotypes.

Transformed seeds containing the 326::653656 construct were sown underconditions described in the High Throughput Screening—T1 Generationprotocol. Multiple seedlings/events appeared much larger than thecontrol, but exhibited no apparent negative phenotypes, such as reducedrosette size or seed yield, as mature plants.

EXAMPLE 9 Lead ME20023; Genomic Locus At1g226945 (SEQ ID NO: 80)

Plant Construct Generation Stage Assay Result 35S::At1g26945 T₁ plantsSeedling Morphological Significant PhenotypingME20023 is Homologous to Leads 80 & 81.

ME20023 was identified from a reciprocal BLAST algorithm as havingapproximately 80% identity to Leads 80 & 81.

Multiple Events of ME21445 Showed Significantly Enhanced Growth as T₁Seedlings, with Elongated Hypocotyls, Flat Inflorescences, and OblongLeaves.

Transformed seeds containing the 35S::At1g26945 construct were sownunder conditions described in the High Throughput Screening—T1Generation protocol. Multiple seedlings/events appeared much larger andwith elongated hypocotyls compared to the control. The plants exhibitedflat inflorescences and oblong leaves at maturity.

EXAMPLE 10 Determination of Functional Homolog Sequences

The “Lead” sequences described in above Examples are utilized toidentify functional homologs of the lead sequences and, together withthose sequences, are utilized to determine a consensus sequence for agiven group of lead and functional homolog sequences.

A subject sequence is considered a functional homolog of a querysequence if the subject and query sequences encode proteins having asimilar function and/or activity. A process known as Reciprocal BLAST(Rivera et al, Proc. Natl Acad. Sci. USA,1998, 95:6239-6244) is used toidentify potential functional homolog sequences from databasesconsisting of all available public and proprietary peptide sequences,including NR from NCBI and peptide translations from Ceres clones.

Before starting a Reciprocal BLAST process, a specific query polypeptideis searched against all peptides from its source species using BLAST inorder to identify polypeptides having sequence identity of 80% orgreater to the query polypeptide and an alignment length of 85% orgreater along the shorter sequence in the alignment. The querypolypeptide and any of the aforementioned identified polypeptides aredesignated as a cluster.

The main Reciprocal BLAST process consists of two rounds of BLASTsearches; forward search and reverse search. In the forward search step,a query polypeptide sequence, “polypeptide A,” from source species S^(A)is BLASTed against all protein sequences from a species of interest. Tophits are determined using an E-value cutoff of 10-5 and an identitycutoff of 35%. Among the top hits, the sequence having the lowestE-value is designated as the best hit, and considered a potentialfunctional homolog. Any other top hit that had a sequence identity of80% or greater to the best hit or to the original query polypeptide isconsidered a potential functional homolog as well. This process isrepeated for all species of interest.

In the reverse search round, the top hits identified in the forwardsearch from all species are used to perform a BLAST search against allprotein or polypeptide sequences from the source species S^(A). A tophit from the forward search that returned a polypeptide from theaforementioned cluster as its best hit is also considered as a potentialfunctional homolog.

Functional homologs are identified by manual inspection of potentialfunctional homolog sequences. Representative functional homologs areshown in FIG. 1. The Figure represents a grouping of a lead/querysequence aligned with the corresponding identified functional homologsubject sequences. Lead sequences and their corresponding functionalhomolog sequences are aligned to identify conserved amino acids and todetermine a consensus sequence that contains a frequently occurringamino acid residue at particular positions in the aligned sequences, asshown in FIG. 1.

Each consensus sequence then is comprised of the identified and numberedconserved regions or domains, with some of the conserved regions beingseparated by one or more amino acid residues, represented by a dash (−),between conserved regions.

Useful polypeptides of the inventions, therefore, include each of thelead and functional homolog sequences shown in FIG. 1, as well as theconsensus sequences shown in the Figure. The invention also encompassesother useful polypeptides constructed based upon the consensus sequenceand the identified conserved regions. Thus, useful polypeptides includethose which comprise one or more of the numbered conserved regions ineach alignment table in FIG. 1, wherein the conserved regions may beseparated by dashes. Useful polypeptides also include those whichcomprise all of the numbered conserved regions in FIG. 1, alternativelycomprising all of the numbered conserved regions in an individualalignment table and in the order as depicted in FIG. 1. Usefulpolypeptides also include those which comprise all of the numberedconserved regions in the alignment table and in the order as depicted inFIG. 1, wherein the conserved regions are separated by dashes, whereineach dash between two adjacent conserved regions is comprised of theamino acids depicted in the alignment table for lead and/or functionalhomolog sequences at the positions which define the particular dash.Such dashes in the consensus sequence can be of a length ranging fromlength of the smallest number of dashes in one of the aligned sequencesup to the length of the highest number of dashes in one of the alignedsequences.

Such useful polypeptides can also have a length (a total number of aminoacid residues) equal to the length identified for a consensus sequenceor of a length ranging from the shortest to the longest sequence in anygiven family of lead and functional homolog sequences identified in FIG.1.

The present invention further encompasses nucleotides that encode theabove described polypeptides, as well as the complements thereof, andincluding alternatives thereof based upon the degeneracy of the geneticcode.

The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the invention can be made. Such modificationsare to be considered within the scope of the invention as defined by thefollowing claims.

TABLE 2 summarizes the sequences found in FIG. 1.

Each of the references from the patent and periodical literature citedherein is hereby expressly incorporated in its entirety by suchcitation. TABLE 2 FUNCTIONAL HOMOLOG ID % IDENTITY E-VALUE SPECIESSEQ_ID_NO QUERY HOMOLOG ID: CeresClone 519 SEQ ID NO 87 663844 76.003.30E−30 Glycine max SEQ ID NO 99 703180 72.70 9.19E−26 Triticumaestivum SEQ ID NO 102 945972 69.80 4.60E−24 Brassica napus SEQ ID NO103 22331645 69.50 3.00E−27 Arabidopsis thaliana SEQ ID NO 106 3143196865.80 1.99E−16 Oryza sativa subsp. japonica SEQ ID NO 109 50912765 58.502.49E−16 Oryza sativa subsp. japonica SEQ ID NO 110 486120 53.571.20E−16 Zea mays SEQ ID NO 111 503296 55.42 2.50E−16 Zea mays SEQ ID NO112 QUERY HOMOLOG ID: CeresClone 4734 SEQ ID NO 85 663844 69.60 2.49E−23Glycine max SEQ ID NO 99 703180 76.60 1.09E−29 Triticum aestivum SEQ IDNO 102 945972 68.60 1.60E−21 Brassica napus SEQ ID NO 103 22331645 64.403.70E−22 Arabidopsis thaliana SEQ ID NO 106 31431968 51.10 1.49E−16Oryza sativa subsp. japonica SEQ ID NO 109 50912765 53.40 2.80E−17 Oryzasativa subsp. japonica SEQ ID NO 110 486120 54.65 1.50E−18 Zea mays SEQID NO 111 503296 50.59 9.40E−17 Zea mays SEQ ID NO 112 QUERY HOMOLOG ID:CeresClone 8607 SEQ ID NO 97 663844 67.40 5.29E−23 Glycine max SEQ ID NO99 703180 75.50 4.79E−29 Triticum aestivum SEQ ID NO 102 945972 68.604.69E−22 Brassica napus SEQ ID NO 103 1449794 73.80 5.69E−26 Populusbalsamifera subsp. trichocarpa SEQ ID NO 107 1468218 67.80 4.09E−23Populus balsamifera subsp. trichocarpa SEQ ID NO 100 1530225 75.002.10E−26 Populus balsamifera subsp. trichocarpa SEQ ID NO 104 2233164563.60 9.79E−22 Arabidopsis thaliana SEQ ID NO 106 31431968 50.602.49E−16 Oryza sativa subsp. japonica SEQ ID NO 109 50912765 52.901.49E−16 Oryza sativa subsp. japonica SEQ ID NO 110 78708592 56.807.09E−19 Oryza sativa subsp. japonica SEQ ID NO 98 486120 48.61 5.40E−14Zea mays SEQ ID NO 111 503296 50.59 9.40E−17 Zea mays SEQ ID NO 112QUERY HOMOLOG ID: CeresClone 336524 SEQ ID NO 83 663844 64.80 1.70E−17Glycine max SEQ ID NO 99 703180 64.90 9.99E−20 Triticum aestivum SEQ IDNO 102 945972 56.50 3.70E−15 Brassica napus SEQ ID NO 102 22331645 54.601.09E−15 Arabidopsis thaliana SEQ ID NO 106 31431968 84.30 1.60E−28Oryza sativa subsp. japonica SEQ ID NO 109 50912765 73.20 3.99E−25 Oryzasativa subsp. japonica SEQ ID NO 110 486120 67.82 1.10E−24 Zea mays SEQID NO 111 503296 77.50 4.40E−26 Zea mays SEQ ID NO 112 QUERY HOMOLOG ID:CeresClone 560681 SEQ ID NO 89 663844 80.60 6.99E−28 Glycine max SEQ IDNO 99 703180 100.00 5.79E−40 Triticum aestivum SEQ ID NO 102 94597273.80 3.99E−25 Brassica napus SEQ ID NO 103 22331645 64.70 4.30E−21Arabidopsis thaliana SEQ ID NO 106 31431968 62.30 1.29E−19 Oryza sativasubsp. japonica SEQ ID NO 109 50912765 60.40 4.90E−20 Oryza sativasubsp. japonica SEQ ID NO 110 486120 59.77 1.20E−18 Zea mays SEQ ID NO111 503296 56.47 2.10E−19 Zea mays SEQ ID NO 112 QUERY HOMOLOG ID:CeresClone 560948 SEQ ID NO 91 663844 77.70 3.90E−27 Glycine max SEQ IDNO 99 703180 89.00 4.39E−35 Triticum aestivum SEQ ID NO 102 945972 71.905.90E−24 Brassica napus SEQ ID NO 103 22331645 64.40 3.30E−21Arabidopsis thaliana SEQ ID NO 106 31431968 60.00 6.39E−18 Oryza sativasubsp. japonica SEQ ID NO 109 50912765 59.30 1.90E−18 Oryza sativasubsp. japonica SEQ ID NO 110 486120 58.62 6.40E−18 Zea mays SEQ ID NO111 503296 56.32 1.30E−17 Zea mays SEQ ID NO 112 QUERY HOMOLOG ID:CeresClone 653656 SEQ ID NO 93 663844 96.70 8.60E−39 Glycine max SEQ IDNO 99 703180 81.80 1.29E−28 Triticum aestivum SEQ ID NO 102 945972 76.808.29E−25 Brassica napus SEQ ID NO 103 1449794 79.50 1.70E−26 Populusbalsamifera subsp. trichocarpa SEQ ID NO 107 1468218 87.90 6.40E−34Populus balsamifera subsp. trichocarpa SEQ ID NO 100 1530225 80.608.10E−27 Populus balsamifera subsp. trichocarpa SEQ ID NO 104 2233164572.50 1.29E−26 Arabidopsis thaliana SEQ ID NO 106 31431968 64.108.20E−18 Oryza sativa subsp. japonica SEQ ID NO 109 50912765 60.705.00E−18 Oryza sativa subsp. japonica SEQ ID NO 110 78708592 73.801.49E−25 Oryza sativa subsp. japonica SEQ ID NO 98 486120 60.87 8.70E−14Zea mays SEQ ID NO 111 503296 59.04 9.10E−19 Zea mays SEQ ID NO 112QUERY HOMOLOG ID: CeresClone 733804 SEQ ID NO 95 663844 78.80 1.49E−27Glycine max SEQ ID NO 99 703180 72.20 4.39E−26 Triticum aestivum SEQ IDNO 102 945972 71.70 1.39E−24 Brassica napus SEQ ID NO 103 1449794 70.001.70E−24 Populus balsamifera subsp. trichocarpa SEQ ID NO 107 146821874.40 3.00E−27 Populus balsamifera subsp. trichocarpa SEQ ID NO 1001530225 71.10 6.50E−25 Populus balsamifera subsp. trichocarpa SEQ ID NO104 22331645 71.10 3.60E−24 Arabidopsis thaliana SEQ ID NO 106 3143196864.20 8.79E−21 Oryza sativa subsp. japonica SEQ ID NO 109 50912765 62.704.90E−20 Oryza sativa subsp. japonica SEQ ID NO 110 78708592 82.202.50E−32 Oryza sativa subsp. japonica SEQ ID NO 98 486120 57.75 9.70E−15Zea mays SEQ ID NO 111 503296 57.47 1.70E−19 Zea mays SEQ ID NO 112

REFERENCES

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1. An isolated nucleic acid molecule comprising: (a) a nucleotidesequence encoding an amino acid sequence that is at least 85% identicalto any one of Leads 80, 81, 113, 114, ME08328, ME01905, ME21445, andME20023, SEQ ID NOS. 94, 96, 90, 82, 88, 84, 86, 92 and 80,respectively; (b) a nucleotide sequence that is complementary to any oneof the nucleotide sequences according to paragraph (a); (c) a nucleotidesequence according to any one of SEQ ID NOS. 94, 96, 90, 82, 88, 84, 86,92 and 80; (d) a nucleotide sequence that is in reverse order of any oneof the nucleotide sequences according to (c) when read in the 5′ to 3,direction; (e) a nucleotide sequence that is an interfering RNA to thenucleotide sequence according to paragraph (a); (f) a nucleotidesequence able to form a hybridized nucleic acid duplex with the nucleicacid according to any one of paragraphs (a)-(d) at a temperature fromabout 40° C. to about 48° C. below a melting temperature of thehybridized nucleic acid duplex; (f) a nucleotide sequence encoding anyone of the amino acid sequences identified as Leads 80, 81, 113, 114,ME08328, ME01905, ME21445, and ME20023, corresponding to SEQ ID NOS. 95,97, 91, 83, 89, 85, 87, 93 and 81, respectively; or (g) a nucleotidesequence encoding any one of the lead, functional homolog or consensussequences in FIG.
 1. 2. A vector, comprising: a) a first nucleic acidhaving a regulatory region encoding a plant transcription and/ortranslation signal; and a second nucleic acid having a nucleotidesequence according to any one the nucleotide sequences of claim 1,wherein said first and second nucleic acids are operably linked.
 3. Amethod of modulating plant size, modulating vegetative growth,modulating plant architecture, seedling vigor and/or modulating theplant biomass, said method comprising introducing into a plant cell anisolated nucleic acid comprising a nucleotide sequence selected from thegroup consisting of: (a) a nucleotide sequence encoding an amino acidsequence that is at least 85% identical to any one of Leads 80, 81, 113,114, ME08328, ME01905, ME21445, and ME20023, SEQ ID NOS. 94, 96, 90, 82,88, 84, 86, 92 and 80, respectively; (b) a nucleotide sequence that iscomplementary to any one of the nucleotide sequences according toparagraph (a); (c) a nucleotide sequence according to any one of SEQ IDNOS. 94, 96, 90, 82, 88, 84, 86, 92 and 80; (d) a nucleotide sequencethat is in reverse order of any one of the nucleotide sequencesaccording to (c) when read in the 5′ to 3′ direction; (e) a nucleotidesequence that is an interfering RNA to the nucleotide sequence accordingto paragraph (a); (f) a nucleotide sequence able to form a hybridizednucleic acid duplex with the nucleic acid according to any one ofparagraphs (a)-(d) at a temperature from about 40° C. to about 48° C.below a melting temperature of the hybridized nucleic acid duplex; (f) anucleotide sequence encoding any one of the amino acid sequencesidentified as Leads 80, 81, 113, 114, ME08328, ME01905, ME21445, andME20023, corresponding to SEQ ID NOS. 95, 97, 91, 83, 89, 85, 87, 93 and81, respectively; or (g) a nucleotide sequence encoding any one of thelead, functional homolog or consensus sequences in FIG. 1, wherein saidplant produced from said plant cell has modulated plant size, modulatedvegetative growth, modulated plant architecture, modulated seeding vigorand/or modulated biomass as compared to the corresponding level intissue of a control plant that does not comprise said nucleic acid. 4.The method according to claim 3, wherein said consensus sequencecomprises one or more of the conserved regions identified in any one ofthe alignment tables in FIG.
 1. 5. The method according to claim 4,wherein said consensus sequence comprises all of the conserved regionsidentified in FIG.
 1. 6. The method according to claim 5, wherein saidconsensus sequence comprises all of the conserved regions and in theorder identified in FIG.
 1. 7. The method according to claim 6, whereinsaid conserved regions are separated by one or more amino acid residues.8. The method according to claim 7, wherein each of said of one or moreamino acids consisting in number and kind of the amino acids depicted inthe alignment table for the lead and/or functional homolog sequences atthe corresponding positions that.
 9. The method according to claim 8,wherein said consensus sequence has a length in terms of total number ofamino acids that is equal to the length identified for a consensussequence in FIG. 1, or equal to a length ranging from the shortest tothe longest sequence in FIG.
 1. 10. The method of claim 3, wherein saiddifference is an increase in the level of plant size, vegetative growth,organ number, seedling vigor and/or biomass.
 11. The method of claim 3,wherein said isolated nucleic acid is operably linked to a regulatoryregion.
 12. The method of claim 11, wherein said regulatory region is apromoter selected from the group consisting of YP0092 (SEQ ID NO: 38),PT0676 (SEQ ID NO: 12), PT0708 (SEQ ID NO: 17), PT0613 (SEQ ID NO: 5),PT0672 (SEQ ID NO: 11), PT0678 (SEQ ID NO: 13), PT0688 (SEQ ID NO: 15),PT0837 (SEQ ID NO: 24), the napin promoter, the Arcelin-5 promoter, thephaseolin gene promoter, the soybean trypsin inhibitor promoter, the ACPpromoter, the stearoyl-ACP desaturase gene, the soybean α′ subunit ofβ-conglycinin promoter, the oleosin promoter, the 15 kD zein promoter,the 16 kD zein promoter, the 19 kD zein promoter, the 22 kD zeinpromoter, the 27 kD zein promoter, the Osgt-1 promoter, the beta-amylasegene promoter, and the barley hordein gene promoter.
 13. The method ofclaim 11, wherein said regulatory region is a promoter selected from thegroup consisting of p326 (SEQ ID NO: 76), YP0144 (SEQ ID NO: 55), YP0190(SEQ ID NO: 59), p13879 (SEQ ID NO: 75), YP0050 (SEQ ID NO: 35), p32449(SEQ ID NO: 77), 21876 (SEQ ID NO: 1), YP0158 (SEQ ID NO: 57), YP0214(SEQ ID NO: 61), YP0380 (SEQ ID NO: 70), PT0848 (SEQ ID NO: 26), andPT0633 (SEQ ID NO:7), the cauliflower mosaic virus (CaMV) 35S promoter,the mannopine synthase (MAS) promoter, the 1′ or 2′ promoters derivedfrom T-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34Spromoter, actin promoters such as the rice actin promoter, and ubiquitinpromoters such as the maize ubiquitin-1 promoter.
 14. The method ofclaim 11, wherein said regulatory region is a promoter selected from thegroup consisting of ribulose-1,5-bisphosphate carboxylase (RbcS)promoters such as the RbcS promoter from eastern larch (Larix laricina),the pine cab6 promoter, the Cab-1 gene promoter from wheat, the CAB-1promoter from spinach, the cab1R promoter from rice, the pyruvateorthophosphate dikinase (PPDK) promoter from corn, the tobacco Lhcb1*2promoter, the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter,and thylakoid membrane protein promoters from spinach (psaD, psaF, psaE,PC, FNR, atpC, atpD, cab, rbcS, PT0535 (SEQ ID NO: 3), PT0668 (SEQ IDNO: 2), PT0886 (SEQ ID NO: 29), PR0924 (SEQ ID NO: 78), YP0144 (SEQ IDNO: 55), YP0380 (SEQ ID NO: 70) and PT0585 (SEQ ID NO: 4).
 15. A plantcell comprising an isolated nucleic acid comprising a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence encoding an amino acid sequence that is at least 85% identicalto any one of Leads 80, 81, 113, 114, ME08328, ME01905, ME21445, andME20023, corresponding to SEQ ID NOS. 94, 96, 90, 82, 88, 84, 86, 92 and80, respectively; (b) a nucleotide sequence that is complementary to anyone of the nucleotide sequences according to paragraph (a); (c) anucleotide sequence according to any one of SEQ ID NOS. 94, 96, 90, 82,88, 84, 86, 92 and 80; (d) a nucleotide sequence that is in reverseorder of any one of the nucleotide sequences according to (c) when readin the 5′ to 3′ direction; (e) a nucleotide sequence that is aninterfering RNA to the nucleotide sequence according to paragraph (a);(f) a nucleotide sequence able to form a hybridized nucleic acid duplexwith the nucleic acid according to any one of paragraphs (a)-(d) at atemperature from about 40° C. to about 48° C. below a meltingtemperature of the hybridized nucleic acid duplex; (f) a nucleotidesequence encoding any one of the amino acid sequences identified asLeads 80, 81, 113, 114, ME08328, ME01905, ME21445, and ME20023corresponding to SEQ ID NOS. 95, 97, 91, 83, 89, 85, 87, 93 and 81,respectively; or (g) a nucleotide sequence encoding any one of the lead,functional homolog or consensus sequences in FIG.
 1. 16. A transgenicplant comprising the plant cell of claim
 15. 17. Progeny of the plant ofclaim 16, wherein said progeny has modulated plant size, modulatedvegetative growth, modulated plant architecture, modulated seedlingvigor and/or modulated biomass as compared to the corresponding level intissue of a control plant that does not comprise said nucleic acid. 18.Seed from a transgenic plant according to claim
 16. 19. Vegetativetissue from a transgenic plant according to claim
 16. 20. A food productcomprising vegetative tissue from a transgenic plant according to claim16.
 21. A feed product comprising vegetative tissue from a transgenicplant according to claim
 16. 22. A product comprising vegetative tissuefrom a transgenic plant according to claim 16 used for the conversioninto fuel or cherpical feedstocks.
 23. A method for detecting a nucleicacid in a sample, comprising: providing an isolated nucleic acidaccording to claim 1; contacting said isolated nucleic acid with asample under conditions that permit a comparison of the nucleotidesequence of the isolated nucleic acid with a nucleotide sequence ofnucleic acid in the sample; and analyzing the comparison.
 24. A methodfor promoting increased biomass in a plant, comprising: (a) transforminga plant with a nucleic acid molecule comprising a nucleotide sequenceencoding any one of the lead, functional homolog or consensus sequencesin FIG. 1; and (b) expressing said nucleotide sequence in saidtransformed plant, whereby said transformed plant has an increasedbiomass or enhance seedling vigor as compared to a plant that has notbeen transformed with said nucleotide sequence.
 25. A method formodulating the biomass of a plant, said method comprising altering thelevel of expression in said plant of a nucleic acid molecule accordingto claim 1.